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2,405 | https://bio-protocol.org/exchange/protocoldetail?id=2405&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Assessing Plant Tolerance to Acute Heat Stress
MK Minsoo Kim
FM Fionn McLoughlin
EB Eman Basha
EV Elizabeth Vierling
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2405 Views: 11367
Edited by: Marisa Rosa
Reviewed by: Magdalena Migocka
Original Research Article:
The authors used this protocol in Oct 2016
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Oct 2016
Abstract
It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth. Here, we provide protocols for assays that can be used quantitatively or qualitatively to assess the relative ability of plants to acquire tolerance to high temperature stress. The hypocotyl elongation assay described was developed to screen for mutants defective in the acquisition of tolerance to extreme temperature stress, and other assays were developed to further characterize mutant and transgenic plants for heat tolerance of other processes or at other growth stages. Although the protocols provide details for application to Arabidopsis thaliana, the same basic methods can be adopted to assay heat tolerance in other plant species.
Keywords: Thermotolerance Greening Arabidopsis Heat shock proteins
Background
It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth, and many studies have identified genes necessary for, or associated with temperature acclimation. Typically, acclimation requires a period of exposure to a non-damaging temperature treatment, either above optimal to induce heat tolerance, or below optimal to induce cold or freezing tolerance. In Arabidopsis and other plants, freezing tolerance can be achieved within a period of 24 h of cold treatment (cold hardening), with maximum freezing tolerance occurring after several days of hardening (Gilmour et al., 1988; Thomashow, 1999). Tolerance to normally lethal or damaging high temperatures can develop more rapidly, within a few hours. This type of rapid heat acclimation was extensively documented by Yarwood (1967) and others in the 1960s using a variety of plant species. Research on high temperature acclimation accelerated when it was recognized in the early 1980s that all organisms, including plants, responded to heat stress with a ‘heat shock response’ that involves transcription and translation of a conserved set of ‘heat shock proteins’ (Lindquist, 1986; Vierling, 1991). Work with soybean seedlings, similar to the earlier work of Yarwood, defined a simple assay for hypocotyl elongation that allowed investigation of the relationship of heat acclimation to the heat shock response (Lin et al., 1984). This assay was then extended to Arabidopsis and used to identify mutants altered in heat acclimation, in both forward and reverse genetic screens (Hong and Vierling, 2001; Larkindale et al., 2005; Kim et al., 2012). Other assays for heat acclimation were also developed and used for mutant screening in Arabidopsis, including heat acclimation of greening of dark grown seedlings (Burke et al., 2000) and seedling viability (Wu et al., 2013).
Because plants can experience rapid daily temperature cycles, it is perhaps not surprising that heat tolerance can be acquired on a time scale consistent with diurnal changes in temperature, and that acclimation treatments afford maximal protection if they are administered 24 h or less before the imposition of damaging heat stress. It is also important to recognize that plant responses to temperature vary significantly with the length and severity, as well as the developmental timing of the temperature treatment. A review by Yeh et al. (2012) provides an excellent description of how variations in temperature treatments can affect phenotypic outcomes. Here we describe in detail basic methods that have been used to assess the ability of plants to acclimate to severe high temperature using Arabidopsis thaliana. Done precisely, these assays can provide quantitative information on the relative heat tolerance of different plant genotypes or of plants grown under different conditions. They can also readily be developed to use with other plant species.
Materials and Reagents
100 x 15 mm square Petri dish with grid (Simport, catalog number: D210-16 )
Any brand sterile pipette tips that fit pipettors in 3 above
Parafilm (Bemis, catalog number: PM996 )
Aluminum foil (any brand)
100 x 15 mm round Petri dish (Fisher Scientific, catalog number: S33580A )
Filter paper
Arabidopsis thaliana Columbia-0 seeds, hot1-3 mutant seeds (ABRC, catalog number: CS16284 )
Household Bleach (Clorox or any brand that contains 5.25% sodium hypochlorite, 6 month shelf-life when stored at room temperature)
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
MS basal salt mixture (Sigma-Aldrich, catalog number: M5524 ), store at 4 °C
2-(N-Morpholino) ethanesulfonic acid (MES) hydrate (Sigma-Aldrich, catalog number: M8250 )
Phyto agar (Sigma-Aldrich, catalog number: A1296 )
Sucrose (Fisher Scientific, catalog number: S5-500 )
Potassium hydroxide (KOH) (Fisher Scientific, catalog number: P250-500 )
Seed sterilization solution (see Recipes)
Half-strength MS agar media (see Recipes)
Equipment
Any brand of pipettors to dispense 2 to 20 μl, 20 to 100 μl, and 0.1 to 1.0 ml (e.g., Eppendorf, catalog numbers: 4924000037 , 4924000061 , 3123000063 ; or comparable)
Growth chamber (Percival Scientific, model: AR41L2 )
Oven incubator (Precision, model: Thelco incubator 3DM , catalog number: 51221120)
Sharp pen
Scale loupe (CWJ, Peak, model: 1983 10x Scale Loupe )
Dissecting scope (Leica Microsystems, model: Leica MZ6 or comparable)
Leveling table (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: H18310-0000 )
1 L glass bottles (Fisher Scientific)
Autoclave
Software
Microsoft Excel
ImageJ (optional)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Kim, M., McLoughlin, F., Basha, E. and Vierling, E. (2017). Assessing Plant Tolerance to Acute Heat Stress. Bio-protocol 7(14): e2405. DOI: 10.21769/BioProtoc.2405.
McLoughlin, F., Basha, E., Fowler, M. E., Kim, M., Bordowitz, J., Katiyar-Agarwal, S. and Vierling, E. (2016). Class I and II Small Heat Shock Proteins Together with HSP101 Protect Protein Translation Factors during Heat Stress. Plant Physiol 172(2): 1221-1236.
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Category
Plant Science > Plant physiology > Abiotic stress
Plant Science > Plant physiology > Plant growth
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2,406 | https://bio-protocol.org/exchange/protocoldetail?id=2406&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus
ES Eleonora Sendersky
RS Ryan Simkovsky
SG Susan S. Golden
RS Rakefet Schwarz
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2406 Views: 10134
Edited by: Dennis Nürnberg
Reviewed by: Maria SinetovaAlexander Martin Ruecker
Original Research Article:
The authors used this protocol in Aug 2016
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Abstract
A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells.
Keywords: Biofilm Cyanobacteria Synechococcus elongatus Chlorophyll measurement Sessile Planktonic
Background
Several recently published studies indicate an emerging interest in the mechanisms that underlie cell-aggregation and biofilm development in cyanobacteria (Fisher et al., 2013; Jittawuttipoka et al., 2013; Schatz et al., 2013; Enomoto et al., 2014; Schwarzkopf et al., 2014; Enomoto et al., 2015; Oliveira et al., 2015; Agostoni et al., 2016; Parnasa et al., 2016). We recently reported a self-biofilm-inhibitory mechanism that dictates planktonic growth of the model unicellular cyanobacterium Synechococcus elongatus PCC 7942 (Schatz et al., 2013; Nagar and Schwarz, 2015). Abrogation of the biofilm-inhibitory process by inactivation of particular genes results in robust biofilm development in this otherwise planktonic strain (Schatz et al., 2013; Nagar and Schwarz, 2015). These studies required quantification of the extent of biofilm development in various strains and under different conditions. Crystal violet is commonly used for quantification of biofilms in heterotrophic bacteria (O’Toole and Kolter, 1998). This staining procedure, however, quantifies only the sessile fraction of cells. Here we provide a detailed protocol for culture growth and quantification of cyanobacterial biofilms using chlorophyll measurement as a proxy for biomass accumulation in sessile as well as in planktonic cells. These measurements allow estimation of the total biomass accumulated and representation of the relative fraction of chlorophyll in sessile or in planktonic cells.
Materials and Reagents
Custom-made Pyrex glass tubes for bacterial liquid cultures (200 x 32 mm, made from Pyrex tubing, Corning, catalog number: 8510-32-D)
Note: Can order from http://www.degroot.co.il/.
‘Sponge plug’ (Plastic foam stoppers, 27 x 34 mm) (Jaece Industies, catalog number: L800-C )
Pasteur pipettes (230 mm) (Romical, catalog number: 94-08401002 )
0.45 µm syringe filter (Sartorius, catalog number: 16555 )
Filter Stericup-GP 250 ml Express Plus PES (0.22 μm) (EMD Millipore, catalog number: SCGPU05RE )
Silicone tubing (6 x 9 mm, 4 x 6 mm) (Degania Silicone, catalog numbers: 2110600234 , 2110400434 , respectively)
Sterile pipettes 1 and 25 ml
1 ml pipettes (Corning, Costar®, catalog number: 4011 )
25 ml pipettes (Corning, Costar®, catalog number: 4489)
Sterilized pipette tips (Corning, Axygen®, catalog numbers: T-200-C , T-1000-C )
Eppendorf tubes (1.5 ml) (Corning, Axygen®, catalog number: MCT-175-C )
Synechococcus elongatus PCC7942
Sodium nitrate (NaNO3) (Sigma-Aldrich, catalog number: S8170 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: K26364082)
Note: This product has been discontinued. Alternatively, Merck, catalog number: 105886 can be used.
Calcium chloride dihydrate (CaCl2·2H2O) (ICN, catalog number: 10035-04-8)
Note: This product has been discontinued. Alternatively, Bio-Lab, catalog number: 034205 can be used.
Potassium phosphate dibasic (K2HPO4) (Honeywell International, Riedel-de-Haen, catalog number: 04248 )
Ethylenediaminetetraacetic acid (Na2Mg·EDTA) (Bio-Lab, catalog number: 05142359)
Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 051423 can be used.
Ferric ammonium citrate (C6H11FeNO7) (MP Biomedicals, catalog number: 02158040 )
Citric acid (C6H8O7) (Frutarom, catalog number: 2355511000)
Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 030205 can be used.
Boric acid (H3BO3) (Bio-Lab, catalog number: 02010591)
Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 020105 can be used.
Manganese chloride hexahydrate (MnCl2·6H2O) (Duchefa Biochemie, catalog number: M0533 )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (CARLO ERBA Reagents, catalog number: 494907 )
Sodium molybdate dihydrate (Na2MoO4·2H2O) (MP Biomedicals, catalog number: 194863 )
Copper sulphate pentahydrate (CuSO4·5H2O) (Honeywell International, Riedel-de-Haen, catalog number: 12849 )
Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Honeywell International, Riedel-de-Haen, catalog number: 12922)
Note: This product has been discontinued. Alternatively, Sigma-Aldrich, catalog number: 239267 can be used.
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H3375 )
Sodium hydroxide (NaOH) (Frutarom, catalog number: 5553510 )
Acetone ((CH3)2CO), A.C.S. reagent (Avantor Performance Materials, J.T. Baker®, catalog number: 9006-03 )
Sodium bicarbonate (NaHCO3) (DAEJUNG CHEMICAL & METALS, catalog number: 7566-4100 )
Sodium thiosulfate (Na2S2O3) (Sigma-Aldrich, catalog number: 217247 )
Bacto agar (BD, BactoTM, catalog number: 214010 )
LB agar (Lennox) (BD, DifcoTM, catalog number: 240110 )
Antibiotics (added as appropriate according to the resistance of the particular strain)
Spectinomycin dihydrochloride pentahydrate (Duchefa Biochemie, catalog number: S0188 )
Kanamycine sulphate monohydrate (Duchefa Biochemie, catalog number: K0126 )
Gentamycin sulphate (Duchefa Biochemie, catalog number: G0124 )
Chloramphenicol (Duchefa Biochemie, catalog number: C0113 )
Liquid BG11-medium (see Recipes)
Stock I (100x concentrated)
Stock II (100x concentrated)
Stock III (100x concentrated)
Stock V (1,000x concentrated)
Solid BG11-medium (see Recipes)
Equipment
Laminar flow hood
Fume hood
Autoclave
Spectrophotometer (Agilent Technologies, model: Cary 100 , catalog number: 10069000) and respective cuvettes (Cell type P.L = 10 mm/EA) (Starna Cells, catalog number: 9-SOG-10)
Benchtop centrifuge (MiniSpin, max. centrifugal force: 12,100 x g) (Eppendorf, model: MiniSpin® , catalog number: 5452000018)
Refrigerator (4-7 °C)
Growth rooms (30 ± 2 °C and 24 ± 2 °C for liquid cultures and cultures on solid medium, respectively)
Note: Growing cultures on solid medium at 24 °C rather than 30 °C allows maintaining the cultures for longer periods.
Light source
For liquid cultures–incandescent light producing a flux of 20-30 μmol photons m-2 sec-1; Cultures on solid-BG11 are illuminated by fluorescent light (5 μmol photons m-2 sec-1)
Pipet-aid
Single-channel pipettes 200 and 1,000 μl
Bubbling system
Air compressor (Oil free air compressor) (Assouline Compressors, model: vs 204 50 )
Cylinder carbon dioxide (CO2, compr. 99.5%) (Maxima, catalog number: GCDCU2.527)
Flow meters (Tuttnauer company, 0-0.1 L/min, 1-10 L/min)
High-flow air pressure regulator (0-2 PSI) (Marsh Bellofram, model: Type 70 , catalog number: 960-129-000)
Note: To bubble cultures with 3% CO2 in air the following setup is used (Figure 1): CO2 gas is mixed with compressed air using flow meters to yield 3% CO2 in air. This mixture is humidified by bubbling into 1.5 L double distilled water (DDW) in a 2 L bottle to prevent water evaporation during bubbling of the cultures. The outlet from this bottle is passed through an empty 2 L bottle (serving to trap residual liquid) and through an air pressure regulator (200 mbar is appropriate for bubbling of ~300 culture tubes). Bubbling of CO2 enriched air from the latter into multiple cultures is obtained using home-made manifolds constructed from silicone tubing and appropriate T-bar connectors. The gas in each manifold channel is passed through a 0.45 µm filter (Figure 1). A short video is provided to demonstrate the flow rate in individual cultures tubes (Video 1).
Figure 1. A scheme describing the setup used to bubble cultures with CO2-enriched air
Video 1. Cyanobacterial cultures under bubbling
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sendersky, E., Simkovsky, R., Golden, S. S. and Schwarz, R. (2017). Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus. Bio-protocol 7(14): e2406. DOI: 10.21769/BioProtoc.2406.
Download Citation in RIS Format
Category
Microbiology > Microbial biofilm > Biofilm culture
Cell Biology > Cell isolation and culture > Cell growth
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2,407 | https://bio-protocol.org/exchange/protocoldetail?id=2407&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice
Michael A. Schmid
EK Elizabeth Kauffman
AP Anne Payne
EH Eva Harris
LK Laura D. Kramer
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2407 Views: 17688
Edited by: Alka Mehra
Original Research Article:
The authors used this protocol in Jun 2016
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Jun 2016
Abstract
Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between pathogens, mosquito vectors, and the mammalian host. In addition, this protocol can help establish improved infection challenge models for pre-clinical testing of vaccines or therapeutics that take into account the natural route of transmission via mosquitoes.
Keywords: Mosquito Saliva Salivary gland Extract Virus transmission Intradermal Needle Mouse
Background
While probing for blood, the mosquito inoculates saliva that facilitates feeding but can also contain pathogens, if the mosquito has previously fed on an infected individual. Mosquito saliva plays an important role in establishing infection, facilitating dissemination, modulating immune responses, and exacerbating pathogenesis during West Nile virus (Schneider et al., 2006; Styer et al., 2011), Dengue virus (Cox et al., 2012; Conway et al., 2014; McCracken et al., 2014; Schmid et al., 2016), chikungunya virus (Agarwal et al., 2016), Semliki Forest virus (Pingen et al., 2016), Rift Valley Fever virus (Le Coupanec et al., 2013) and Plasmodium parasite (Schneider et al., 2011) infection. Many important questions yet remain and call for improved animal models.
Whereas inoculation via infected mosquitoes best mimics natural transmission, high variability in the inoculated dose and limited availability of insectary facilities result in restricted use of such procedures. In addition, the amount of saliva and the presence or absence of mosquito-derived components cannot be controlled when using infected mosquitoes. As an alternative, ‘spot feeding’ of uninfected female mosquitoes followed by intradermal inoculation of the pathogen via a needle mimics the natural deposition of saliva into mouse skin and delivers a defined dose of pathogen. The ‘spot feeding’ model has successfully been used to study Dengue virus (Cox et al., 2012; McCracken et al., 2014) and West Nile virus (Moser et al., 2015) infection but still requires the concomitant use of live mosquitoes and mice, and cannot control for the amount of saliva delivered. To separately control for mosquito and mouse experiments, inserting the mosquito proboscis into a sucrose solution in a capillary tube can serve to collect mosquito saliva artificially. The saliva collected during sugar feeding, however, differs qualitatively from mosquito saliva that is inoculated into the host skin during natural blood feeding (Marinotti et al., 1990; Moser et al., 2015).
Here, we describe the use of a simplified model of needle-inoculating mosquito salivary gland extract (SGE) from non-infected mosquitoes that can be delivered with a pathogen in a controlled manner at a defined dose. This method allows for independent handling of live mosquitoes and mice between collaborators and can more easily be standardized between assays and research groups. Use of SGE has proven useful to study infection with West Nile virus (Schneider et al., 2006; Moser et al., 2015), Dengue virus (Conway et al., 2014; Schmid et al., 2016), Rift Valley fever virus (Le Coupanec et al., 2013), and Sindbis virus (Schneider et al., 2004). Injection of SGE does not precisely mimic inoculation via the mosquito proboscis and likely contains non-secreted components of salivary glands. Nevertheless, this method allows collection of higher quantities of mosquito-derived factors, contains all secreted proteins, and can also be used in in vitro assays. Overall, the procedures described here should facilitate collaboration between entomologists, immunologists, and researchers studying pathogens of interest.
Materials and Reagents
Note: For the rearing of mosquitoes and general maintenance of a mosquito colony, see Bio-protocol Kauffman et al. (2017).
Cotton-tipped swab, 15 cm handle (Puritan, catalog number: 25-826 5WC )
Petri dish, sterile, 60 mm (e.g., 60 mm TC-Treated Cell Culture Dish, Corning, Falcon®, catalog number: 353002 )
Plastic wrap
50-ml conical centrifuge tubes (CELLTREAT Scientific Products, catalog number: 229422 )
15-ml conical centrifuge tubes (CELLTREAT Scientific Products, catalog number: 229412 )
5-ml tubes, 12 x 75 mm (Corning, Falcon®, catalog number: 352054 )
96-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 15041 )
5-ml serological pipets (CELLTREAT Scientific Products, catalog number: 229206B )
10-ml serological pipets (CELLTREAT Scientific Products, catalog number: 229211B )
Microcentrifuge tubes, 1.5 ml (Corning , Axygen®, catalog number: MCT-150-C )
Needle: 30-gauge, small RN hub, custom-made, point style 4 (10°-12° bevel), 25-mm length (Hamilton, catalog number: 7803-07 )
Note: Alternative needles: use with disposable hypodermic needle, e.g., 30 G x 1 inch (BD, catalog number: 305128 ).
Frosted microscope slides, 25 x 75 x 1.0 mm (e.g., Fisher Scientific, catalog number: 12-550-343 )
Wooden applicator sticks, 15 cm (Puritan, catalog number: 807 )
Insect Pins Morpho Black enameled No.000 (BioQuip, catalog number: 1208B000 )
Dissecting probes fabricated from wooden applicator sticks and insect pins
Note: Dissecting probes are fabricated from 15-cm wooden applicators and insect pins. Soak the applicator sticks in hot water for at least 30 min, and cut the heads off the pins and discard. Using plyers, hold the pin, avoiding damage to the pointy end, and push the blunt (cut) end into the stick. Let the probe dry.
Transfer pipets, 1 ml, bulk, nonsterile (BioLogix, catalog number: 30-0135 )
pH test strips (e.g., Sigma-Aldrich, catalog number: P4536-100EA )
Female mosquitoes
Mosquito holding carton (see Bio-protocol Kauffman et al., 2017 for description and construction)
Mice
Triethylamine (Sigma-Aldrich, catalog number: T0866 )
Ethyl alcohol
Phosphate-buffered saline (PBS), low endotoxin ≤ 0.25 EU/ml (i.e., Mediatech, catalog number: 21-030-CV )
Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23235 )
Endpoint Chromogenic Limulus Amebocyte Lysate QCL-1000 Assay (Lonza, catalog number: 50-647U )
Disinfectant, such as Bleach or Umonium38 (Huckert’s Laboratoire International, catalog number: PF 12209 )
Isoflurane, Iso-Vet (Chanelle, catalog number: CDS019936 ) for anesthesia of mice
70% ethanol: Combine 74 ml of ethyl alcohol with 26 ml of water. Label as flammable and store at room temperature for up to 3 months
Alexa Fluor 680
Monoclonal antibody 4G2
Equipment
Stir plate and stir bar
Floating microtube rack (i.e., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5974-4015 )
Forceps, Superfine Tips, Swiss Style #5 (BioQuip, catalog number: 4535 )
Cover slip forceps (Fisher Scientific, catalog number: S17328C )
Manufacturer: Medco Instruments, catalog number: S17328C .
Note: It is important that the flat parts of the forceps touch each other over the entire surface. If the opposing surfaces touch at only one point, the ear skin is more likely to generate folds when inserting the needle.
Stereoscopic Zoom Microscope (e.g., Nikon Instruments, model: SMZ1500 )
Branson Sonifier Model 450 (Branson 450 Analog Sonifier with 1/2” Horn, 400 W, 120 VAC) (Cole-Parmer, catalog number: EW-04715-03 )
Manufacturer: Emerson Electric, BRANSON, model: Model 450 .
Cup Horn for Ultrasonic Processors, 3” dia (Cole-Parmer, catalog number: EW-04715-39)
Manufacturer: Emerson Electric, BRANSON, catalog number: 109-116-1760 .
Branson Sound-Proof Enclosure for Ultrasonic Processors (Cole-Parmer, catalog number: EW-04715-44)
Manufacturer: Emerson Electric, BRANSON, catalog number: 101-063-275 .
Refrigerated centrifuge (e.g., Eppendorf, model: 5417 R )
Reusable glass microinjection syringe type 702RN, no needle, volume 25 µl (Hamilton, catalog number: 7636-01 )
Note: Alternative syringe: Reusable glass microinjection syringe model 702 LT (Luer tip), SYR, NDL sold separately, no needle, volume 25 µl (Hamilton, catalog number: 80401 ).
Heat block capable of heating to 37 °C and 60 °C (e.g., Digital Dry Block Heater, Single Block, 240 V, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88870004 )
Plate reader capable of reading absorption at 562 nm and 405-410 nm, and temperature control capable of heating to 37 °C (e.g., BioTek Instruments, model: ELx808 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Schmid, M. A., Kauffman, E., Payne, A., Harris, E. and Kramer, L. D. (2017). Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice. Bio-protocol 7(14): e2407. DOI: 10.21769/BioProtoc.2407.
Download Citation in RIS Format
Category
Immunology > Animal model > Mouse
Microbiology > Microbe-host interactions > In vivo model
Cell Biology > Tissue analysis > Tissue isolation
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2,408 | https://bio-protocol.org/exchange/protocoldetail?id=2408&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Isolation of Fucus serratus Gametes and Cultivation of the Zygotes
AS Amandine Siméon
Cécile Hervé
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2408 Views: 7830
Reviewed by: Rebecca Van Acker
Original Research Article:
The authors used this protocol in Nov 2016
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Abstract
Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually mature fronds in the laboratory and thousands of synchronously developing zygotes are easily obtained. In contrast to other eukaryotic models, such as land plants (Brownlee and Berger, 1995), the embryo is free of maternal tissues and therefore readily amenable to pharmacological approaches. The zygotes are relatively large (up to 100 µm in diameter), facilitating manipulations and imaging studies. During the first hours of zygote development, the alignment of the axis to external cues such as light is labile and can be reversed by light gradients from different directions. A few hours before rhizoid emergence, the alignment of the axis and the polarity are fixed and the cells germinate accordingly. At this stage the zygotes are naturally attached to the substratum through the secretion of cell wall adhesive materials (Kropf et al., 1988; Hervé et al., 2016). The first cell division occurs about 24 h after fertilisation and the early embryo is composed of only two cell types that differ in size, shape and developmental fates (i.e., thallus cells and rhizoid cells) (Bouget et al., 1998). The embryo can be successfully cultivated in the laboratory for a few more days (4 weeks maximum) and has an invariant division pattern during the early stages, which allows cell lineages to be traced histologically.
Keywords: Asymmetric cell division Developmental biology Brown algae Fucus serratus Zygotes Embryogenesis
Background
This protocol provides instructions for the isolation of male and female gametes of Fucus spp. used for in vitro fertilisation and discusses how to monitor the development of the resulting zygotes and early embryos. These instructions are for the use of the dioecious species Fucus serratus, but they can be readily adapted to a monoecious Fucale species.
Materials and Reagents
Razor blade (Gilette)
Black flat base
Note: This can be a black paper base.
Paper towel (Lucart Professional, catalog number: 864043 )
100 μm nylon filter (Sigma-Aldrich, catalog number: NY1H00010 )
Slides
Petri dishes (SARSTEDT, catalog number: 82.1472.001 )
Sexually mature plants of Fucus serratus, males and females
Note: Marine Research Institutes are sometimes equipped with facilities that allow the ordering and sending of such material (see for instance the EMBRC-France website, http://www.embrc-france.fr/en) The algae can be transported in moist tissues for a time preferentially no longer than 2 days.
Approximately 1 L of natural seawater
Note: Alternatively artificial seawater can be prepared or purchased (Sigma-Aldrich, catalog number: S9883 ).
Equipment
Beaker of approximately 250 ml (Fisher scientific)
Desk lamp
Fridge or cold room
Optical microscope equipped with 20x and/or 40x objectives (Olympus)
Thermostatic chamber at 13 °C (Pol-Eko Aparatura)
White light, ideally 90 µmol m-2 sec-1 (Mazda)
Hermetic black chamber (optional)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Siméon, A. and Hervé, C. (2017). Isolation of Fucus serratus Gametes and Cultivation of the Zygotes. Bio-protocol 7(14): e2408. DOI: 10.21769/BioProtoc.2408.
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Category
Plant Science > Plant cell biology > Cell isolation
Cell Biology > Cell isolation and culture > Co-culture
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2,409 | https://bio-protocol.org/exchange/protocoldetail?id=2409&type=0 | # Bio-Protocol Content
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Rice Lamina Joint Inclination Assay
HL Hsing-Yi Li*
HW Hsin-Mei Wang*
Seonghoe Jang
*Contributed equally to this work
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2409 Views: 11824
Edited by: Marisa Rosa
Reviewed by: Laia ArmengotYuko Kurita
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina joint inclination bioassays, we applied the protocol presented below. The protocol focuses on: (1) how to prepare rice samples for the assay, and (2) how to treat BL exogenously. Finally, we have added a result showing lamina inclination between WT and osbul1 in BL solutions of various concentrations.
Keywords: Bioassay Brassinosteroid Lamina inclination Lamina joint Rice
Background
The rice lamina joint connects the leaf blade and sheath, contributing significantly to the leaf angle trait and BR is the main regulator of the trait, while other plant hormones, including ethylene, gibberellin, and auxin, also influence leaf angle (Gan et al., 2015). A more erect leaf facilitates the penetration of sunlight, enhancing photosynthetic efficiency and occupying less space in dense planting (Sakamoto et al., 2006). Thus, rice lamina inclination is one of the major agronomic traits affecting rice plant architecture. Actually, the rice lamina inclination assay developed mainly by Wada and his co-workers is a highly specific and sensitive bioassay for BRs (Wada et al., 1981 and 1984). In this bioassay, treatment with BRs induces greater cell expansion of adaxial cells relative to the abaxial cells in the joint regions, causing laminar inclination in a concentration-dependent manner (Takeno and Pharis, 1982; Cao and Chen, 1995). Changes in cell wall extensibility or loosening are essential for cell expansion (Campbell and Braam, 1999). Although the molecular mechanism for such action remains elusive, cell wall loosening enzymes including xyloglucan endotransglycosylase have been shown to be upregulated by BL and involved in this modification, resulting in laminar inclination in rice (Uozu et al., 2000). Thus, here we describe a procedure through which we could distinguish the BR sensitivity between the wild type and erect leafed osbul1 mutant plants through the rice lamina inclination assay.
Materials and Reagents
250 µl pipette tips (Mettler-Toledo International, Rainin, catalog number: 17007479 )
1 ml pipette tips (Mettler-Toledo International, Rainin, catalog number: 17001121 )
50 ml SuperClear centrifuge tube (Labcon, catalog number: LAB3181 )
Filter paper (Advantec, No.1: 90 mm)
Petri dish, round, 90 x 15 mm (Alpha Plus, catalog number: 16001-1 )
50 ml syringe (Sigma-Aldrich, catalog number: Z124990 )
Syringe filter (VWR, catalog number: 89041-306 )
Micropore tape (3M, catalog number: 1530-0 )
1 ml tubes
Rice seeds: Orysa sativa spp. japonica cv. Hwayoung and OsBUL1 knockout mutant rice (osbul1)
Ethanol (Avantor Performance Materials, J.T. Baker®, catalog number: 8006 )
Sodium hypochlorite (NaOCl, Commercial Bleach–CLOROX)
Tween 20 (Alfa Aesar, Affymetrix/USB, catalog number: J20605 )
Potassium hydroxide (KOH) (SHOWA, catalog number: 1637-0150 )
Murashige & Skoog basal medium with Vitamins (MS) (PhytoTechnology Laboratories, catalog number: M519 )
Sucrose (Alfa Aesar, Affymetrix/USB, catalog number: J21938 )
Phytogel (Sigma-Aldrich, catalog number: P8169-500G )
Brassinolide (BL) (Sigma-Aldrich, catalog number: E1641 )
Sodium hypochlorite solution (with final available chlorine of 2%) (see Recipes)
5 N potassium hydroxide (KOH) solution (see Recipes)
Murashige & Skoog (MS) media (see Recipes)
1 mM Brassinolide (BL) stock solution (see Recipes)
Equipment
Rice husker (KETT ELECTRIC LABORATORY, model: TR-130 )
Ultrasonic cleaner (Elma, model: E-30H )
Clean bench (Chu-An, model: MBH-420N )
Scissors (Basic Life, catalog number: 76000 )
Forceps (Basic Life, catalog number: BL6502 )
Growth chamber (CHANG KUANG, model: CK-68EX )
Digital camera (Sony, model: NEX-3N )
Protractor (Taiwan united stationery, catalog nunber: HA401 )
600 ml beaker (DWK Life Sciences, DURAN, catalog number: 21 106 48 )
Glass petri dish (Sun Chion, catalog number: B16A1-0090 )
Autoclave
10 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 08 04 )
100 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 24 02 )
500 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 44 03 )
Vortex mixer (Vortex-Genie2, Scientific Industries, model: Model G560 )
Incubator (YIHDER TECHNOLOGY, model: LM-570RD )
Pipetmans (Gilson, models: P20 , P200 and P1000 )
RiOsTM Essential 16 Water Purification System (EMD Millipore, model: RiOsTM Essential 16 )
Summit Series Analytical Balance (Denver Instrument, model: SI-234 )
pH meter (UltraBasic Benchtop pH Meter, Denver Instrument, model: UB-10 )
Software
ImageJ (https://imagej.nih.gov/ij/) for lamina angle measurement
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Li, H., Wang, H. and Jang, S. (2017). Rice Lamina Joint Inclination Assay. Bio-protocol 7(14): e2409. DOI: 10.21769/BioProtoc.2409.
Jang, S., An, G. and Li, H. Y. (2017). Rice Leaf Angle and Grain Size Are Affected by the OsBUL1 Transcriptional Activator Complex. Plant Physiol 173(1): 688-702.
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Category
Plant Science > Plant physiology > Phenotyping
Plant Science > Plant biochemistry > Plant hormone
Biochemistry > Other compound > Plant hormone
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241 | https://bio-protocol.org/exchange/protocoldetail?id=241&type=0 | # Bio-Protocol Content
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Synthesis of 5’ end-labeled RNA
Harald Putzer
Published: Vol 2, Iss 14, Jul 20, 2012
DOI: 10.21769/BioProtoc.241 Views: 13393
Original Research Article:
The authors used this protocol in Dec 2011
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Abstract
5’ end-labeled RNA molecules are useful substrates to analyse the endo- and exonucleolytic activities of various ribonucleases. Here two protocols are given to synthesize P32 labeled RNAs with a 5’ PPP or 5’ P moiety. 5’ exoribonucleases generally do not work on 5’ PPP RNA and require a 5’ P substrate. The activity of certain endoribonucleases like Escherichia coli (E. coli) RNase E or Bacillus subtilis (B. subtilis) RNase Y can be stimulated by a 5’ P moiety.
Keywords: RNA Labeling RNA Synthesis Radioactive labeling
Materials and Reagents
RQ1 DNase (Promega Corporation, catalog number: M6101 )
Calf intestinal phosphatase (F. Hoffmann-La Roche, catalog number: 713023 )
Phenol/Chlorophorm (1:1) (MP Biomedicals, catalog number: AQUAPH01 )/
(Carlo Erba, catalog number: 438601 )
T7 RNA polymérase (Promega Corporation, catalog number: P207B )
T4 Polynucleotide Kinase (Biolabs, catalog number: M0201 )
RNasin RNase inhibitor (Promega Corporation, catalog number: N2611 )
DTT (Promega Corporation, catalog number: P117B )
NTPs (F. Hoffmann-La Roche, catalog number: set1277057 )
γ32P GTP (6,000 Ci/mmole, 10 μC/μl) (PerkinElmer, catalog number: BLU504Z )
γ32P ATP (3,000 Ci/mmole, 10 μCi/μl) (PerkinElmer, catalog number: BLU502A )
3 M NaOAc (pH 4.7) (see Recipes)
Equipment
IllustraTM MicroSpinTM G-25 column (GE Healthcare, model: 27-5325-01 )
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Putzer, H. (2012). Synthesis of 5’ end-labeled RNA. Bio-protocol 2(14): e241. DOI: 10.21769/BioProtoc.241.
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Category
Molecular Biology > RNA > RNA synthesis
Molecular Biology > RNA > RNA labeling
Molecular Biology > RNA > Transcription
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2,410 | https://bio-protocol.org/exchange/protocoldetail?id=2410&type=0 | # Bio-Protocol Content
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Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC
Sheo Shankar Pandey
Prashantee Singh
Biswajit Samal
Raj Kumar Verma
SC Subhadeep Chatterjee
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2410 Views: 8656
Edited by: Arsalan Daudi
Reviewed by: Srujana Samhita YadavalliBin Tian
Original Research Article:
The authors used this protocol in Nov 2016
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Abstract
Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.
Keywords: Xanthomonas siderophores Xanthoferrin Bacterial siderophore estimation HPLC Amberlite XAD-16 resin columns
Background
The Xanthomonas group of bacterial phytopathogens possess an xss (Xanthomonas siderophore synthesis) operon, which is required to produce xanthoferrin (an α-hydroxy carboxylate-type siderophore; similar to vibrioferrin) and to encode an outer membrane receptor involved in the siderophore-mediated iron uptake (Pandey and Sonti, 2010; Pandey et al., 2016a and 2016b). Siderophores are small iron-chelating compounds secreted by bacteria to utilize the insoluble form of iron (Neilands, 1995). For the past three decades, CAS (Chrome azurol S)-agar plate assay has been mostly employed to measure bacterial siderophores by monitoring the halo formation around bacterial colonies (Schwyn and Neilands, 1987; Pandey and Sonti, 2010). However, the above assay cannot reliably be used to quantify siderophores from bacteria which also secrete organic acids (e.g., oxalic acid, citric acid, and isocitric acids) along with siderophores, as the organic acids are capable of chelating iron from CAS dye to compromise the accuracy to quantify siderophore (Rai et al., 2015). Since the above mentioned assay only gives the idea about the bacterial siderophore production qualitatively, hence HPLC-mediated siderophore quantification is a better method than the CAS-agar plate assay for bacteria that produce both siderophores along with organic acids; such as Xanthomonas species (Rai et al., 2015; Pandey et al., 2016a and 2016b).
Materials and Reagents
Pipette tips (Corning, Axygen®, catalog numbers: TF-300-R-S , TF-200-R-S , and TF-1000-R-S )
Culture tubes (Borosil, catalog number: 9800U06 )
Petri dishes (Tarson Products, catalog number: 460140-140MM )
Stericup® filter units with 0.22 μm porosity (EMD Millipore, catalog number: SCGPU02RE )
Centrifuge tubes: 500 ml, 50 ml, 15 ml, 2 ml, and 1.5 ml (Tarson Products, Kolkata, India)
Cork borer, 8 ± 0.2 mm (HiMedia Laboratories, catalog number: LA737 )
Syringe driven filter unit with 0.22 μm porosity (Millex®-GS) (EMD Millipore, catalog number: SLGS033SS )
Xanthomonas campestris pv. campestris 8004, X. oryzae pv. oryzicola BXOR1, X. oryzae pv. oryzae (Indian isolate) and ∆xssA mutant in Xanthomonas siderophore synthesis gene A (Lab collections)
Rifampicin
2,2’-dipyridyl (DP) (Sigma-Aldrich, catalog number: 14453 )
Note: This product has been discontinued.
Amberlite XAD16N, 20-60 mesh (Sigma-Aldrich, catalog number: XAD16-1KG )
Methanol (Thermo Fisher Scientific, catalog number: Q32407 )
Standard vibrioferrin (Fujita et al., 2011; see Notes 2 and 3)
Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: T6508-25ML )
Acetonitrile (CH3CN) (Sigma-Aldrich, catalog number: 34888-1L )
Note: This product has been discontinued.
Peptone (HiMedia Laboratories, catalog number: CR001 )
Sucrose (HiMedia Laboratories, catalog number: GRM601-500G )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
Chrome Azural S (CAS) (Merck, catalog number: 1.02477.0025 )
Ferric chloride (FeCl3) (Standard Reagents, catalog number: SRCF005C )
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: 29507 )
Hexadecyltrimethylammonium bromide (HDTMA) (Sigma-Aldrich, catalog number: H6269-250G )
BactoTM agar (BD, BactoTM, catalog number: 214010 )
BactoTM peptone (BD, BactoTM, catalog number: 211677 )
Agar (HiMedia Laboratories, catalog number: RM026-500G )
Peptone-sucrose (PS) medium (Tsuchia et al., 1982; see Recipes)
CAS solution (Schwyn and Neilands, 1987; see Recipes)
Peptone-sucrose-agar (PSA) medium (see Recipes)
PSA-CAS-DP plate (see Recipes)
Equipment
Conical flasks: 500 ml (Vensil Glass Works, catalog number: 1161 )
Conical flasks: 2,000 ml (Borosil, catalog number: 4980030 )
Micro-pipettes (Eppendorf, catalog number: EPPR4396 )
Shaking incubator (Eppendorf, New BrunswickTM, model: Innova® 43 )
SORVALL RC-5B PLUS Superspeed centrifuge (Thermo Fisher Scientific, Waltham, MA, USA)
pH meter (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EC-PH510/11S )
Agilent 1100 series HPLC system (Agilent Technologies, model: Agilent 1100 Series )
Agilent C-18 (4.6 mm x 250 mm x 5 μm) column (Agilent Technologies, Santa Clara, CA, USA)
Burette (BOROSIL, catalog number: 2123016 ), burette support stand and clamps
Vacuum concentrator plus (Eppendorf, model: Concentrator Plus , catalog number: 5305000304)
Millipore vacuum, pressure pump (EMD Millipore, catalog number: XI04 220 50 )
Autoclave
Software
ChemStation Software; Rev. A.10.02 [1757] (Agilent, Santa Clara, CA, USA)
Microsoft Office Excel 2013 (Microsoft Corporation, Redmond, USA)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pandey, S. S., Singh, P., Samal, B., Verma, R. K. and Chatterjee, S. (2017). Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC. Bio-protocol 7(14): e2410. DOI: 10.21769/BioProtoc.2410.
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Category
Microbiology > Microbe-host interactions > Bacterium
Plant Science > Plant immunity > Host-microbe interactions
Biochemistry > Lipid > Extracellular lipids
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2,411 | https://bio-protocol.org/exchange/protocoldetail?id=2411&type=0 | # Bio-Protocol Content
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Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader
MM Magdiel Martínez
NM Namyr A. Martínez
WS Walter I. Silva
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2411 Views: 32119
Edited by: Andrea Puhar
Reviewed by: David A. Cisneros
Original Research Article:
The authors used this protocol in Jun 2016
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Abstract
Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007; Martínez et al., 2016). Changes in intracellular calcium concentration can be measured using the calcium sensitive fluorescent ratiometric dye fura-2 AM. This method is a high throughput way to measure agonist mediated calcium responses.
Keywords: Cell biology Cell based analysis Ion analysis Calcium Mammalian cell line Astrocyte Physiology
Background
Activation of G protein coupled receptors triggers production in hundreds or thousands of second messenger molecules. Once stimulated, Gq protein coupled receptors activate phospholipases C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG remains membrane bound and IP3 is released into the cytosol where it binds to a specific IP3 receptor (calcium channel) in the endoplasmic reticulum (ER). This causes an increase of intracellular calcium concentration that modulates the activation of calcium binding proteins and Protein Kinase C (PKC). Therefore, given the importance of calcium in biological systems, many techniques/methods to analyze and measure Ca2+ levels have been established.
To determine the intracellular Ca2+ concentration, fluorescent indicators are particularly useful. Ca2+ indicators are available with variations in affinity, brightness or spectral characteristics. Fura-2-acetoxymethyl ester (fura-2 AM), is a membrane-permeable, non-invasive derivative of the ratiometric calcium indicator fura-2. Fura-2 AM crosses cell membranes and once inside the cell, the cellular esterases remove the acetoxymethyl group. Ca2+-bound fura-2 AM has its excitation maximum at 335 nm, Ca2+-free fura-2 AM has its excitation maximum at 363 nm. In both states, the emission maximum is about 510 nm. The typical excitation wavelengths used are 340 nm and 380 nm for Ca2+-bound and Ca2+-free fura-2 AM, respectively. The ratios 510 nm/340 nm and 510 nm/380 nm are directly related to the amount of intracellular Ca2+ (Figure 1). Traditionally, changes in intracellular Ca2+ mobilization/concentration were measured using fluorescent calcium indicators and a fluorescent confocal microscope, known as calcium imaging. This protocol explains how to use the Tecan Infinite M200® microplate reader equipped with an injector to measure intracellular Ca2+ concentration. With the aid of an injector equipped microplate reader, researchers may be able to screen new agonist for Gq protein couple receptors and evaluate how the intracellular calcium mobilization is affected by mutant receptors. This protocol can be adapted to measure agonist mediated intracellular calcium mobilization in several cellular/receptor/agonist models using the appropriate growth conditions.
Figure 1. The human P2Y2 receptor mobilizes intracellular Ca2+ efficiently when expressed in 1321N1 human astrocytoma cells. Representative traces of intracellular calcium responses to 100 μM ATP in 1321N1 cells transfected with hHA-P2Y2R (solid squares) and control non-transfected 1321N1 cells (solid circles). Data represent the mean ± SEM of readings from 4 independent experiments.
Materials and Reagents
15 ml centrifuge tube (VWR, catalog number: 89004-368 )
Clear flat-bottom black 96-well culture trays with lid (Corning, catalog number: 3904 )
Wild type (WT) human 1321N1 astrocytoma cells devoid of P2 receptors and hHA-P2Y2R expressing 1321N1 cells (gift from Dr. Gary A. Weisman, University of Missouri)
DPBS
Trypsin (TrypLE Express) (Thermo Fisher Scientific, GibcoTM, catalog number: 12604013 )
Probenecid (water soluble) (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36400 )
Adenosine 5’-triphosphate disodium salt (ATP) (Tocris Bioscience, catalog number: 3245 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
HEPES (Sigma-Aldrich, catalog number: H0887 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
Fura-2-acetoxymethyl ester (fura-2 AM) (Thermo Fisher Scientific, InvitrogenTM, catalog number: F1225 )
Pluronic F-127, 20% (w/v) stock in DMSO (Thermo Fisher Scientific, InvitrogenTM, catalog number: P3000MP )
Dulbecco’s modified Eagle’s medium–low glucose (DMEM) (Sigma-Aldrich, catalog number: D2902 )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178 )
Antibiotic/antimicotic (Sigma-Aldrich, catalog number: A5955 )
Geneticin, G418 (Thermo Fisher Scientific, GibcoTM, catalog number: 10131035 )
HEPES buffered saline (HBS), pH 7.4 (see Recipes)
Dye solution (see Recipes)
Medium for wild type (WT) human 1321N1 astrocytoma cells (see Recipes)
Medium for hHA-P2Y2R expressing 1321N1 cells (see Recipes)
Low serum medium (see Recipes)
Equipment
Tecan Infinite M200® (Tecan Trading, model: Infinite® M200 )
Fisher Scientific Mini vortex (Fisher Scientific)
Eppendorf 5810R Benchtop centrifuge (Eppendorf, model: 5810 R )
Biorad TC10 cell counter® (Bio-Rad Laboratories, model: TC10TM Automated Cell Counter )
Note: This product has been discontinued.
Fisher Scientific Isotemp CO2 Incubator (5% CO2, 37 °C) (Fisher Scientific)
Laminar flow biological hood
Software
i-Control® (Tecan Trading AG, Switzerland)
Excel® (Microsoft)
GraphPad Prism® (GraphPad Software Inc., La Jolla, CA)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Martínez, M., Martínez, N. A. and Silva, W. I. (2017). Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader. Bio-protocol 7(14): e2411. DOI: 10.21769/BioProtoc.2411.
Martínez, N. A., Ayala, A. M., Martínez, M., Martínez-Rivera, F. J., Miranda, J. D. and Silva, W. I. (2016). Caveolin-1 regulates the P2Y2 receptor signaling in human 1321N1 astrocytoma cells. J Biol Chem 291(23): 12208-12222.
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Category
Cancer Biology > General technique > Cell biology assays
Neuroscience > Cellular mechanisms > Intracellular signalling
Cell Biology > Cell signaling > Intracellular Signaling
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2,412 | https://bio-protocol.org/exchange/protocoldetail?id=2412&type=0 | # Bio-Protocol Content
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Peer-reviewed
Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue
JM Jeremy John Mathan
SI Salim Ismail
JM Jennifer Jane McGhee
TS Trevor Sherwin
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2412 Views: 6967
Reviewed by: Federica Pisano
Original Research Article:
The authors used this protocol in Jun 2016
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The authors used this protocol in:
Jun 2016
Abstract
Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue (Mathan et al., 2016). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human corneal tissue. This protocol describes the procedure for sphere formation and culture, preparation of tissue for sphere implantation, corneal limbus microsurgery and sphere implantation.
Keywords: Cell Culture Cornea Sphere-forming cells Implantation Limbus
Background
Previous research has focused on isolation of limbal cells which were exclusively epithelial (limbal stem cell) or stromal (keratocyte progenitor cell) in order to decipher their individual roles in corneal homeostasis and wound repair. This protocol aims to isolate limbal cells by their functional ability to form spheres in culture and which by their very nature will include a diversity of cells both epithelial and stromal which contribute to the formation of the limbal niche. Subsequent to isolation of these spheres we are investigating their potential use in corneal restoration after implantation. Here we describe an in-vitro surgical protocol for the implantation of these spheres into human corneal tissue and the downstream analysis.
Materials and Reagents
Scalpel blade (ProSciTech, profile 11) (Swann Morton, catalog number: LSB11 )
20 x 100 mm cell culture dish (Corning, Falcon®, catalog number: 353003 )
Cotton buds (Cotton Tips double ended, Protec Solutions, catalog number: 941001690389 )
Transfer pipette (3 ml) (Interlab, catalog number: KJ622-1A )
5 ml tubes (Techno Plas, catalog number: P5016UL )
40 µm strainer (Corning, Falcon®, catalog number: 352340 )
Glass coverslips (covergalss #1 30 mm diam.) (PST, catalog number: G430 )
6-well tissue culture plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
35 x 10 mm cell culture dish (Corning, Falcon®, catalog number: 353001 )
Fresh and frozen human cadaveric donor corneoscleral tissue obtained post-surgery from the New Zealand National Eye Bank
70% ethanol (EMD Millipore, catalog number: 1009832500 )
Phosphate buffered saline (PBS) sterile (Sigma-Aldrich, catalog number: P4417-100TAB )
Phosphate buffered saline and 10% glycerol (AnalaR NORMAPUR) (VWR, catalog number: 24388.320 )
Dispase (Thermo Fisher Scientific, GibcoTM, catalog number: 17105041 )
Collagenase (Blend Type L) (Sigma-Aldrich, catalog number: C8176 )
Hyaluronidase (Sigma-Aldrich, catalog number: H3506 )
Neurobasal-A Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888022 )
Human Epidermal Growth Factor (EGF) (PeproTech, catalog number: AF-100-15 )
Human Fibroblastic Growth Factor (FGF) Basic (PeproTech, catalog number: 100-18B )
B-27 Supplement, 50x (Thermo Fisher Scientific, GibcoTM, catalog number: 12587010 )
N-2 Supplement, 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 17502048 )
GlutaMAX, 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
MEM with GlutaMAX (Minimum Essential Medium) (Thermo Fisher Scientific, GibcoTM, catalog number: 41090036 )
Foetal bovine serum (New Zealand origin) (Thermo Fisher Scientific, GibcoTM, catalog number: 10091148 )
Antibiotic/Antimycotic 100x (Anti/Anti) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
Supplemented Neurobasal-A medium (see Recipes)
Standard culture medium (see Recipes)
Equipment
Tissue culture hood (Heal Force, model: HFsafe 1800 )
Straight scissors (World Precision Instruments, catalog number: 500216 )
Fine forceps (World Precision Instruments, catalog number: 14142 )
Orbital shaker (Ratek Instruments, model: MM1 )
Centrifuge (Sigma Laborzentrifugen, model: 3-15 )
Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HereausTM HeraCell 150 )
Laminar flow cabinet (Gelman, model: HLF 120 )
Binocular Stereo microscope (Carl Zeiss, catalog number: 474110-9904 )
Biological safety cabinet (Email Air Handling, catalog number: 1687-2340-618-3 )
2-20 µl Eppendorf® Pipette (Eppendorf, catalog number: 3120000038 )
Feather MicroScalpel (pfm medical, catalog number: 200300715 )
Crescent Bevel Up Ophthalmic Knife 2.3 mm (MANI, catalog number: MCU26 )
NIKON Digital sight DS-UI camera (Nikon Instruments, model: DS-UI )
Leica DMIL inverted contrasting microscope (Leica Microsystems, model: Leica DMIL )
Leica DM-RA upright fluorescence microscope (Leica Microsystems, model: Leica DM-RA )
Software
NIS-Elements Br Microscope Imaging Software version 3.0
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mathan, J. J., Ismail, S., McGhee, J. J. and Sherwin, T. (2017). Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue. Bio-protocol 7(14): e2412. DOI: 10.21769/BioProtoc.2412.
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Category
Stem Cell > Adult stem cell > Cell transplantation
Cell Biology > Cell isolation and culture > 3D cell culture
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2,413 | https://bio-protocol.org/exchange/protocoldetail?id=2413&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs)
WX Wei Xu
Chengbiao Wu
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2413 Views: 6108
Edited by: Jia Li
Original Research Article:
The authors used this protocol in May 2016
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Abstract
The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) are believed to be involved in many disorders of the brain such as Alzheimer’s disease (AD), schizophrenia, sleep disorders and drug abuse (Mobley et al., 1986). Primary cultures of BFCNs will provide an important tool for studying the mechanism of these diseases. This protocol provides a detailed description of experimental procedures in establishing in vitro primary culture of rat embryonic BFCNs.
Keywords: Basal forebrain cholinergic neuron Nerve growth factor Alzheimer’s disease in vitro culture Microfluidic chamber Axonal transport Neurodegeneration
Background
The basal forebrain cholinergic system innervates the cerebral cortex and hippocampus. The normal function of the BFCNs is essential for normal sleeping, learning and memory. And the atrophy of BFCNs is considered as the early event of Alzheimer’s disease. Thus, the primary BFCNs culture will be the ideal cell model for AD research. In previous studies, primary BFCNs cultures were rarely used. Here, we present a reliable method to isolate and culture BFCNs from the embryonic rat septum which is simple, less time consuming than the previous method (Schnitzler et al., 2008). Our method will greatly facilitate studies of many critical aspects of BFCN function and cell biology.
Materials and Reagents
Pipette tips
Coverglasses (Thermo Fisher Scientific or VWR)
Falcon tubes (50 ml)
Falcon tubes (15 ml)
Sterile pipettes 5, 10, 25 ml
12-well culture plate
10, 15 cm cell culture dishes
Razor blades
Fire-polished glass Pasteur pipettes
Note: Materials #3-9 can be from various suppliers.
Microfluidic chamber (XONA MICROFLUIDICS, catalog number: SND450 )
Stericup® filter units (150, 250 ml) (EMD Millipore, catalog number: SCVPU02RE , 0.10 µm, polyethersulfone)
Pregnant female Sprague Dawley rats (Embryos dissected at E17.5) (Harlan Sprague Dawley)
Poly-L-lysine 0.1% (w/v) in dH2O (Sigma-Aldrich, catalog number: P8920 )
Phosphate-buffered saline (PBS) (without CaCl2, MgCl2) (Thermo Fisher Scientific, GibcoTM, catalog number: 1419094 )
70% ethanol
10x, 2.5% trypsin, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
10x DNase I (10 mg/ml) (Roche Diagnostics, catalog number: 10104159001 )
Anti-TrkA antibody
10x Hanks’ balanced salt solution (HBSS) w/o PR, sod.bicarb, CaMg (Mediatech, catalog number: 20-023-CV )
Penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Neurobasal-A medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888022 )
Fetal bovine serum (FBS) (Qmega Scientific, catalog number: FB-02 or various suppliers) (heat in activation before use)
50x B27 supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
100x GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
Mouse purified NGF as previously published (Mobley et al., 1986)
Dissection buffer (see Recipes)
Plating media (see Recipes)
Maintenance media (see Recipes)
Equipment
Sterile cell culture hood (Esco Micro, model: Class II Type A2 )
Scissors
#5 biologie forces and small spring scissors (Fine Science Tools)
Pipette
Water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: PrecisionTM Model 281 )
Note: This product has been discontinued.
Cell culture incubator (Panasonic, model: MCO-19AIC UV-PA )
Dissecting microscope (Leica, model: Leica S8 AP0 )
Cell culture centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: CL2 )
Autoclave (STERIS, model: AMSCO® Evolution® Steam Sterilizer )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Xu, W. and Wu, C. (2017). Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs). Bio-protocol 7(14): e2413. DOI: 10.21769/BioProtoc.2413.
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Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Cell isolation and culture > Cell isolation
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2,414 | https://bio-protocol.org/exchange/protocoldetail?id=2414&type=0 | # Bio-Protocol Content
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Peer-reviewed
Determination of Survival of Wildtype and Mutant Escherichia coli in Soil
YS Yinka Somorin
CO Conor O'Byrne
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2414 Views: 7794
Reviewed by: Esteban Paredes-Osses
Original Research Article:
The authors used this protocol in Aug 2016
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Abstract
E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival was unknown. This soil survival assay protocol was developed and used to determine the role of the general stress response regulator, RpoS, in the survival of E. coli in soil. Using this soil survival assay, we demonstrated that RpoS was important for the survival of E. coli in soil. This protocol describes the development of the soil survival assay especially the recovery of E. coli inoculated into soil and can be adapted to allow further investigations into the survival of other bacteria in soil.
Keywords: Soil survival Escherichia coli RpoS Environmental persistence Bacteria recovery
Background
Escherichia coli is a Gram-negative, facultative anaerobe, belonging to the Enterobacteriaceae family, which inhabits the intestinal tract of humans, warm-blooded animals and reptiles (Berg, 1996; Gordon and Cowling, 2003). It can be transferred through water and sediments via faeces and is used as an indicator of faecal contamination in drinking and recreational water. The use of E. coli as a faecal indicator is based, at least in part, on the assumption that it exists transiently outside of the host gastrointestinal tract (Ishii and Sadowsky, 2008) and does not survive for a long time in the external environment. However, several studies have isolated E. coli from various natural environments such as municipal wastewater, freshwater, beach water, beach sand and soils (Jiménez et al., 1989; Brennan et al., 2010; Chiang et al., 2011; Byappanahalli et al., 2012; Zhi et al., 2016). The capacity of these E. coli strains to survive for long periods of time and grow in the external environment raises questions about the validity of its continued use as indicator of water quality (Brennan et al., 2010). To understand the genetic mechanism underlying the survival and persistence of E. coli in soil, we developed a soil survival assay to evaluate the role of the different genetic factors on soil survival. We investigated the role of the general stress response regulator, RpoS, in the survival of long-term soil persistent E. coli in soil. The ability of the rpoS mutant (COB583ΔrpoS) to survive in soil was compared with the wildtype strain (COB583) and RpoS was demonstrated to be important for the survival and long-term persistence of E. coli in soil (Somorin et al., 2016). Here, we present the detailed protocol for the soil survival assay and the recovery of E. coli from soil.
Materials and Reagents
Note: All reagents used were from the specified manufacturers (catalog numbers indicated). Nonetheless, the same reagents from different manufacturers are expected to produce similar results.
Spatula
Weighing boat (Sparks Lab Supplies, catalog number: BAL1822 )
Gloves (Sparks Lab Supplies, catalog number: SAF5534X20 )
Sterile micropipette tips (100-1,000 µl) (Abdos Labtech, catalog number: P10102 )
Sterile micropipette tips (2-200 µl) (Abdos Labtech, catalog number: P10101 )
Microcentrifuge tube (1.5 ml) (Abdos Labtech, catalog number: P10202 )
Centrifuge tube (15 ml graduated) (Abdos Labtech, catalog number: P10402 )
Cuvette (LP ITALIANA, catalog number: 112117 )
2 mm laboratory test sieve (B5410/1986, Endecotts, catalog number: 200SIW2.00 )
96-well plate (TC microwell 96U) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 163320 )
90 mm Petri dishes (Abdos Labtech, catalog number: P10901 )
Silty-Loam soil
E. coli strains
E. coli COB583 (Somorin et al., 2016)
E. coli COB583ΔrpoS (Somorin et al., 2016)
Note: E. coli stock cultures were made in LB with 7% Dimethyl sulfoxide (v/v; Sigma-Aldrich) and kept at -80 °C pending use.
Luria-Bertani (LB) broth (Sigma-Aldrich, catalog number: L3022-1KG )
Agar No. 2 (Lab M, catalog number: MC006 )
MacConkey agar (Sigma-Aldrich, catalog number: M7408 )
Distilled water (Milli-Q) (EMD Millipore)
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-100TAB )
LB broth (see Recipes)
LB agar plates (see Recipes)
PBS buffer (see Recipes)
MacConkey agar plates (see Recipes)
Equipment
250 ml conical flask (VWR, catalog number: 214-1132 )
-80 °C freezer
Styrofoam 15 ml tube holder
Bunsen burner
Orbital shaker (Gallenkamp)
Discovery comfort multichannel pipette (20-200 µl; HTL)
Discovery comfort multichannel pipette (5-20 µl; HTL)
Nichipet EX pipette (200-1,000 µl; Nichiryo)
Nichipet EX Pipetman classic pipettes (20-200 µl; Nichiryo)
Vortex mixer (Reax top; Heidolph)
Biomate 3 spectrophotometer (Thermo Fisher Scientific, catalog number: 335904 )
Note: This product has been discontinued.
Weighing scale (Sartorius, catalog number: BL120S )
Note: This product has been discontinued.
Centrifuge (Eppendorf, model: 5418 )
Labo autoclave (SANYO, model: MLS-3020U )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Somorin, Y. and O'Byrne, C. P. (2017). Determination of Survival of Wildtype and Mutant Escherichia coli in Soil. Bio-protocol 7(14): e2414. DOI: 10.21769/BioProtoc.2414.
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Category
Microbiology > Microbial physiology > Adaptation
Microbiology > Microbial cell biology > Cell viability
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2,415 | https://bio-protocol.org/exchange/protocoldetail?id=2415&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae
HH Huanhuan Hao
JH Jing Huang
TL Tongtong Liu
HT Hui Tang
LZ Liping Zhang
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2415 Views: 12876
Edited by: Daan C. Swarts
Reviewed by: Kabin Xie
Original Research Article:
The authors used this protocol in Jul 2016
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Jul 2016
Abstract
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very versatile with the ability to simultaneously target multiple genes within a cell (Varshney et al., 2015). We have developed a CRISPR/Cas9 system to delete large genomic fragments (exceeding 30 kb) in Saccharomyces cerevisiae. One application of this technology is to study the effects of large-scale deletions of non-essential genes which may give insight into the function of gene clusters within chromosomes at the molecular level. In this protocol, we describe the general procedures for large fragment deletion in S. cerevisiae using CRISPR/Cas9 including: how to design CRISPR arrays and how to construct Cas9-crRNA expression plasmids as well as how to detect mutations introduced by the system within S. cerevisiae cells.
Keywords: CRISPR/Cas9 system Large fragment deletion Saccharomyces cerevisiae
Background
The CRISPR/Cas9 system is a rapid, efficient, low-cost, and versatile method for genome editing that can be applied in the fields of biology, agriculture, and medicine. To date, several protocols have been reported on how to make large-scale deletions within genomes. Each of these methods contains its own unique characteristics and advantages. The recently developed CRISPR/Cas9 system for excising large stretches of chromosomes has potential advantages over other methods such as the Latour system (Hirashima et al., 2006). The CRISPR/Cas9 system requires two components: (1) the Cas9 endonuclease for DNA cleavage and (2) a variable guide RNA (gRNA) that directs the Cas9 enzyme in a DNA sequence-specific manner (Cong et al., 2013). When Cas9 is targeted to a genomic locus by a gRNA, Cas9 initiates a DSB. The cell will respond to the DSB by repairing the damage via one of two major pathways: high-fidelity homology-dependent repair or error-prone non-homologous end joining (NHEJ).
The CRISPR/Cas9 system described here requires four components: Cas9 endonuclease, CRISPR array, trans-activating crRNA (tracrRNA), and RNase III (this activity is present in the host cell). The CRISPR array is a genomic locus from which pre-crRNAs are transcribed. In this system, the CRISPR array, engineered on the pCRCT plasmid, expresses multiple spacers flanked by direct repeats driven by a single promoter. Cas9 cannot be targeted by crRNA alone, it requires a crRNA-tracrRNA duplex to target it to a specific site within the genome. Two DNA oligonucleotides that encode for spacer sequences interspaced by a direct repeat (DR) were directly synthesized. The formed dsDNA encoding the crRNA was cloned into a Cas9 expression vector. Once the desired plasmid is constructed, transform S. cerevisiae with it and screen the transformants to obtain mutants with large genomic fragment deletions. In this protocol, the deletion efficiency (10%) is lower than described for deletion of genomic fragments using CRISPR/Cas9 in rice (Zhou et al., 2014). There are two possible reasons. The first one is that the genome may be repaired more rapidly in S. cerevisiae in comparison with that in rice. Another one is a stronger selection pressure used for screening rice transformants. In rice, the selection marker for transformants is hygromycin B, while in yeast Uracil as a selection marker is employed. The stronger selection pressure possibly increase the plasmid copy numbers in rice cells.
Materials and Reagents
Escherichia coli: DH5ɑ (sanyou Biopharmaceuticals)
S. cerevisiae strain: W303 (MATa ura3-52)
pCRCT plasmid (Addgene, catalog number: 60621 )
Salmon DNA (Sigma-Aldrich, catalog number: D1626 )
Restriction enzyme: 10 U/μl Bsal (Takara Bio)
10x buffer G (Takara Bio)
Taq DNA polymerase (Takara Bio)
Antibiotic: 100 µg/ml ampicillin (Siyao)
Pure plasmid mini kit (CWBIO)
Yeast Gen DNA Kit (CWBIO)
DNA oligonucleotide primers (GENEWIZ)
PEG: Poly (ethylene glycol), BioXtra avg. molecular weight 3,350 (Sigma-Aldrich, catalog number: P3640 )
Note: This product has been discontinued.
Yeast extract (Oxoid, catalog number: LP0021 )
Tryptone (Oxoid, catalog number: LP0042 )
Dextrose
Agar (Solarbio, catalog number: A8190 )
Sodium chloride (NaCl) (Tianjin Kemiou Chemical Reagent)
Peptone
Primers
CasYZF: 5’---ACGCTGTAGAAGTGAAAGTTGG---3’
CasYZR: 5’---TAGTATGCTGTGCTTGGGTGTT---3’
Glucose (Tianjin Kemiou Chemical Reagent)
Adenine (Sigma-Aldrich)
Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883-1KG )
Agarose gel recovery kit (Biomiga)
YPD liquid media (see Recipes)
LB plates (with appropriate antibiotics included) (see Recipes)
YPDA liquid medium (see Recipes)
SC medium without uracil (see Recipes)
Equipment
PCR machine (or similar) (Biometra, model: TPfofessional )
42 °C water bath (or similar) (XINBAO, catalog number: HH-501BS )
DNA electrophoresis apparatus (or similar) (SIM International, model: BIO-PRO 200E , catalog number: 0401RHSI049)
Microcentrifuge (SCILOGEX, model: D2012 )
Incubator (or similar, capable of incubation of agar plates at 37 °C or 28 °C) (CIMO, model: DNP-III )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hao, H., Huang, J., Liu, T., Tang, H. and zhang, L. (2017). Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae. Bio-protocol 7(14): e2415. DOI: 10.21769/BioProtoc.2415.
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Category
Microbiology > Microbial genetics > Mutagenesis
Microbiology > Microbial genetics > DNA
Cell Biology > Cell engineering > CRISPR-cas9
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2,416 | https://bio-protocol.org/exchange/protocoldetail?id=2416&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro AMPylation Assays Using Purified, Recombinant Proteins
MT Matthias C. Truttmann
HP Hidde L. Ploegh
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2416 Views: 7003
Edited by: Peichuan Zhang
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation–which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine–has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation (fic) domain-containing AMPylases sparked new interest in this PTM (Kinch et al., 2009; Yarbrough et al., 2009). Based on recent in vivo and in vitro studies, we now know that secreted bacterial AMPylases covalently attach AMP to members of the Rho family of GTPases, while metazoan AMPylases modify HSP70 family proteins in the cytoplasm and the endoplasmic reticulum (ER) (Itzen et al., 2011; Hedberg and Itzen, 2015; Truttmann and Ploegh, 2017). AMPylation is thought to trap HSP70 in a primed yet transiently disabled state that cannot participate in protein refolding reactions (Preissler et al., 2015). In vitro AMPylation experiments are key to assess the activity, kinetics and specificity of protein AMPylation catalyzed by pro- and eukaryotic enzymes. These simple assays require recombinant AMPylases, target proteins (Rho GTPases, HSP70s), as well as ATP as a nucleotide source. Here, we describe strategies to qualitatively and quantitatively study protein AMPylation in vitro.
Keywords: AMPylation Adenylylation Grp78/BiP HSP70 GTPase Proteostasis
Background
Metazoan cell signaling is complex and requires tight control. Aberrations in this well-balanced system threaten cellular homeostasis and induce several maintenance systems aimed at restoring the balance (Kim et al., 2013). Protein AMPylation is directly linked to cellular stress: AMPylation of Rho GTPases by bacterial toxins rewires GTPase-dependent signaling, eventually leading to a collapse of the actin cytoskeleton and cell death (Yarbrough et al., 2009; Mattoo et al., 2011). In contrast, AMPylation of Grp78/BiP in the ER keeps this chaperone in a primed, yet silent conformation to be awoken and set in motion once the burden of unfolded protein in the ER surpasses a certain threshold (Preissler et al., 2015; Sanyal et al., 2015). We and others have extensively used in vitro AMPylation assays to study general properties, target selectivity as well as reaction kinetics of Fic domain-containing AMPylases. We used a combination of distinct in vitro AMPylation assays to i) identify novel targets in complex cell lysates, ii) validate suspected targets and iii) approach the role of AMPylase dimerization and auto-modification as prerequisites for enzymatic activity (Truttmann et al., 2015; 2016 and 2017). Our efforts aim at understanding the scope and impact of the AMPylome on cellular signaling. The in vitro AMPylation assays described herein present methods to achieve this goal.
Materials and Reagents
1.5 ml tubes (1.5 ml Snaplock Microcentrifuge Tube) (Corning, Axygen®, catalog number: MCT-150-C-S )
Pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 9400327 , 9401255 , 9401410 )
Microcentrifuge Tube Locks (Sorenson BioScience, catalog number: 11870 )
Autoradiography film (Carestream Health X-OmatTM LS Film) (Eastman Kodak, catalog number: 05-728-45 )
Saran wrap (generic)
Whatman 3MM filter paper (GE Healthcare, catalog number: 3030-6185 )
Sterile, deionized water (generic)
Ice in isolated containment (generic)
Ethanol (Sigma-Aldrich, catalog number: 362808 )
Purified recombinant AMPylase at 1.0 μg/μl or higher (HIS6-HYPEaa187-437; homemade; see Truttmann et al., 2015)
Purified recombinant target proteins at 1.0 μg/μl or higher (i.e., HIS6-Histone H3; homemade; see Truttmann et al., 2015)
Appropriate TRIS-glycine gels (CriterionTM TGXTM Precast Midi Protein Gel) (Bio-Rad Laboratories, catalog number: 5671023 )
Molecular weight marker (Precision PlusTM Protein Dual Color Standard) (Bio-Rad Laboratories, catalog number: 1610374 )
2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris-base) (Sigma-Aldrich, catalog number: 252859 )
11.8 M hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
[Alpha-33P]ATP, 10 mCi/ml; 3,000 Ci/mmol (Hartman Analytic, catalog number: SRF-207 )
Note: It is of uttermost importance to use [Alpha-33P]ATP and not [Gamma-33P]ATP, which is used to study kinase-dependent phosphorylation events.
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
Potassium chloride (KCl) (EMD Millipore, catalog number: PX1405 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 74255 )
2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
Poly-Phenyl-Oxazole (PPO) (Sigma-Aldrich, catalog number: 216984 )
Dimethyl sulfoxide (DMSO) Sigma-Aldrich, catalog number: 276855 )
1 M Tris-HCl (pH 7.5) (see Recipes)
1 M DTT (see Recipes)
1 M MgCl2 (see Recipes)
100 mM ATP (see Recipes)
5 M NaCl (see Recipes)
Protein storage buffer (see Recipes)
Reaction buffer (see Recipes)
SDS-PAGE 6x sample buffer (see Recipes)
DMSO/PPO solution (see Recipes)
Equipment
Pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 4600170 , 4600240 and 4600250 )
-20 °C freezer (generic)
4 °C refrigerator (generic)
Geiger-counter (generic)
Refrigerated tabletop centrifuge for 1.5 ml Eppendorf tubes (Eppendorf, model: 5810 R )
10 μl Hamilton syringe (Hamilton, catalog number: 80075 )
Radiation safety gear and personal protection equipment (generic)
Vacuum gel dryer (Bio-Rad Laboratories, model: Model 583 )
Glass tray (generic; 5 x 10 inches at least)
Timer (Alarm Timer) (Grainger, catalog number: 8RLR2 )
pH meter (Thermo Fisher Scientific, Thermo ScientificTM, model: Orion StarTM A111 )
Balance (Sartorius, model: Cubis® Precision Balance )
Vortex (Vortex-Genie 2 Vortexer) (VWR, catalog number: VWR-VG3 )
SDS-PAGE system (Bio-Rad Laboratories, model: CriterionTM Cell and PowerPacTM Basic Power Supply, catalog number: 1656019 )
Autoradiography cassettes (FisherBiotech Electrophoresis Systems Autoradiography Cassette, 8 x 10 in) (Fisher Scientific, model: FBAC 810)
Note: This product is not available anymore.
Software
Fiji/ImageJ image analysis software (https://fiji.sc/)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Truttmann, M. C. and Ploegh, H. L. (2017). In vitro AMPylation Assays Using Purified, Recombinant Proteins. Bio-protocol 7(14): e2416. DOI: 10.21769/BioProtoc.2416.
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Category
Biochemistry > Protein > Modification
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2,417 | https://bio-protocol.org/exchange/protocoldetail?id=2417&type=0 | # Bio-Protocol Content
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Peer-reviewed
Mass Spectrometry-based in vitro Assay to Identify Drugs that Influence Cystine Solubility
Neelanjan Bose
Tiffany Zee
Pankaj Kapahi
Marshall L. Stoller
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2417 Views: 7526
Edited by: Jyotiska Chaudhuri
Reviewed by: Marieta RusevaRakesh Bam
Original Research Article:
The authors used this protocol in Mar 2017
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Abstract
Cystinuria is a rare genetic disorder characterized by recurrent, painful kidney stones, primarily composed of cystine, the dimer of the amino acid cysteine (Sumorok and Goldfarb, 2013). Using a mouse model of cystinuria, we have recently shown that administration of drugs that increase cystine solubility in the urine can be a novel therapeutic strategy for the clinical management of the disease (Zee et al., 2017). There is a large unmet need in the field for developing new drugs for cystinuria. To that end, here we describe a simple in vitro cystine solubility assay that is amenable for screening compounds to identify potential drugs that may influence cystine solubility. The assay includes preparing a supersaturated solution of cystine, incubating this solution with drug(s) of choice, and finally using high pressure liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) to quantify the amount of cystine precipitated under various conditions.
Keywords: Cystinuria Kidney stones Cystine Mass spectrometry Drug screening L-cystine dimethyl ester
Background
Cystinuria is a type of kidney stone disease characterized by a genetic defect in cystine transporters in the proximal tubule of the kidney, resulting in massive increase in cystine load in the urine that precipitate as kidney stones (Sumorok and Goldfarb, 2013). While categorized as a rare genetic disorder (~1/15,000 individuals) (Milliner and Murphy, 1993; Palacin et al., 2001), patients suffering from cystinuria experience excruciating pain from recurrent stone episodes (Dent and Senior, 1955). Unlike other, more common kidney stone types (such as calcium oxalate- or uric acid-based), cystine stones are denser and resistant to extracorporeal shock wave lithotripsy (SWL), requiring patients to undergo multiple emergency room visits and surgical procedures to remove obstructive stones (Mattoo and Goldfarb, 2008). The current drug regimen for cystinuria are geared towards either increasing urinary pH (potassium citrate) or reducing urinary cystine levels (thiol drugs, such as tiopronin), which are generally associated with serious side effects (Koraishy et al., 2013; Sumorok and Goldfarb, 2013; Saravakos et al., 2014). Further, clinical (Becker et al., 2007) and murine model (Zee et al., 2017) studies have found little evidence that these drugs are ultimately effective and long-term patient compliance is poor. Thus, there is an urgent need for developing effective therapies to treat cystinuria. Our recent results show that increasing urinary cystine solubility can be a viable alternative strategy for drug development in cystinuria. Herein, we describe an in vitro cystine solubility assay to identify novel compounds capable of influencing cystine solubility.
Materials and Reagents
Gloves
Lab coat
Culture tubes (16 x 100 mm) (VWR, catalog number: 47729-576 )
4 ml glass vials and caps (13-425 thread finish, borosilicate glass) (VWR, catalog number: 46610-706 )
HPLC autosampler vials and caps (9-425 thread finish) (VWR, catalog number: 89523-482 )
Pipette tips (standard 1,000 µl and 200 µl)
Gel loading pipette tips (VWR, catalog number: 37001-150 )
Joint clips to attach the round bottom flask to Allihn condenser (VWR, catalog number: 89426-304)
Manufacturer: GLASSCOLABS, catalog number: 007.470.05A .
Eppendorf tubes (standard 1.5 ml)
Ammonium acetate (for LC-MS LiChropur®) (EMD Millipore, catalog number: 533004 )
Water (HPLC-grade) (VWR, BDH®, catalog number: BDH23595.400 )
Ammonium hydroxide (28% NH3 in H2O, ≥ 99.99%) (Sigma-Aldrich, catalog number: 338818 )
3,3,3’,3’-d4-L-cystine (98%) (Cambridge Isotope Laboratories, catalog number: DLM-1000-1 )
L-cystine (≥ 98%) (Sigma-Aldrich, catalog number: C8755 )
DL-cystine dimethyl ester dihydrochloride (≥ 95%) (Sigma-Aldrich, catalog number: 857327 )
Acetonitrile (HPLC-grade) (VWR, BDH®, catalog number: BDH83639.400 )
Ammonium acetate solution (see Recipes)
Note: Specific brand and catalog number (as used in our work) have been provided for the materials and reagents listed. However, we do not explicitly endorse any of these brands or products and most items can be obtained from other reputed vendors. Further, most of these product/catalog numbers are based on the current availability in the United States, so researchers in other countries may find it necessary to obtain materials from another vendor.
Equipment
pH meter (standard)
Safety goggles
Chemical safety hood
Glass bottles (standard 500 ml and 1 L)
Glass measuring cylinders (standard 1 L, 250 ml, and 100 ml)
Glass round bottom flask (standard 24/40 ground joint, 100 ml)
Magnetic stir-bar (standard)
Reflux apparatus (standard, consisting of stirring-heating mantle, Allihn condenser with a water jacket and 24/40 junction, running water source, rubber tubing, and clips for attachment). Follow vendor instructions for assembly
Pipettes (standard 1,000 µl and 200 µl)
Vortex (standard)
-20 °C freezer (standard)
4 °C incubator or fridge (standard)
HPLC-MS system:
HPLC: Shimadzu UFLC prominence system (HPLC) (Shimadzu, model: Prominence UFLC ) fitted with following modules: CBM-20A (Communication bus module), DGU-A3 (degasser), two LC-20AD (liquid chromatograph, binary pump), SIL-20AC HT (auto sampler)
HPLC column: Luna® NH2 column (2 x 50 mm, 3 µm, 100 Å) (Phenomenex, catalog number: 00B-4377-B0 )
Mass Spectrometer: AB Sciex 4000 LC-MS/MS mass spectrometer (AB Sciex, model: QTRAP® 4000 ) fitted with a Turbo VTM ion source
Centrifuge (standard, with attachment for 4 ml vials)
Weighing scale (standard)
Note: Standard lab equipment should be used for this assay as indicated. Specific brand and catalog numbers (as used in our work) have been provided for components of the HPLC-MS setup–these can be replaced by equipment from other vendors; arguably any HPLC-MS setup will work with this assay, but may need additional optimization based on vendor-provided instructions.
Software
AB Sciex’s Analyst® v1.6.1 for data acquisition, development of LC method, and optimization of analyte-specific MRM (multiple reaction monitoring) transitions
AB Sciex’s Peakview® v2.1 and Skyline® v3.623 for LC-MS/MS data analysis
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bose, N., Zee, T., Kapahi, P. and Stoller, M. L. (2017). Mass Spectrometry-based in vitro Assay to Identify Drugs that Influence Cystine Solubility. Bio-protocol 7(14): e2417. DOI: 10.21769/BioProtoc.2417.
Download Citation in RIS Format
Category
Biochemistry > Other compound > Amino acid
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2,418 | https://bio-protocol.org/exchange/protocoldetail?id=2418&type=0 | # Bio-Protocol Content
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Peer-reviewed
Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis
LT Liku B Tezera
M Magdalena K Bielecka
PE Paul T Elkington
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2418 Views: 8728
Edited by: Alka Mehra
Reviewed by: Marc-Antoine Sani
Original Research Article:
The authors used this protocol in Jan 2017
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The authors used this protocol in:
Jan 2017
Abstract
Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol describes a 3-D system incorporating mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction in tuberculosis.
Keywords: Bioelectrospray Alginate-based matrices Multicellular 3-D cell culture Tuberculosis Collagen Extracellular matrix
Background
Mycobacterium tuberculosis (Mtb) is a pathogen of global public health importance that causes a mortality of 1.8 million people per year and morbidity of 10 million worldwide (WHO, 2016). Despite substantial investment in research, much greater understanding of the host-pathogen interaction is required to improve prevention and treatment. Currently, the pathogen is becoming increasingly resistant to commonly used drugs, with the emergence of extensively drug-resistant Mtb. One of challenges to the tuberculosis (TB) field is the availability of model systems to interrogate the host-pathogen interaction, as widely used animal models do not fully reflect pathology in humans. Hence, there is an urgent need to complement these animal models by developing a physiologically relevant in vitro environment (Bielecka et al., 2017; Tezera et al., 2017). Mtb is an obligate pathogen of man and so we hypothesized that a model system requires human cells, virulent mycobacteria, 3-dimensional organization, extracellular matrix, longitudinal readouts and the ability to modulate the environment over time.
This protocol describes a physiologically relevant in vitro environment by utilizing human cells, extracellular matrix components and live Mtb using a bioelectrospray model to mimic human Mtb infection. This 3-D model is different from other models by using an extracellular matrix that can be released by de-capsulation so that downstream analysis of cells within the matrix can be performed. Furthermore, this methodology has wide potential applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimens and vaccination approaches.
Materials and Reagents
Female Luer Thread Style Connectors (West Group, catalog number: FTLL210-J1A )
Tissue paper
Note: Sterilize lab tissue paper by autoclaving and keep it sterile until use.
Nozzle for bioelectrosprayer (0.7 φ) (2 pieces) (Nisco Engineering, catalog number: PE-00577 )
Silicon tubes with connector attached at the end (3 pieces) (VWR, catalog number: 228-0705 )
FalconTM 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
75 cm2 flask (CELLSTAR® Cell Culture Flasks, Greiner Bio One International)
SterilinTM 7 ml polystyrene Bijou containers (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 129A )
2 ml Eppendorf tubes
20 ml syringe
5 ml syringe
0.22 µm syringe filter EMD Millipore MillexTM-GP 33mm (Fisher Scientific, catalog number: 10268401)
Manufacturer: EMD Millipore, catalog number: SLGP033RS .
Pipette (10 ml, 25 ml) (CELLSTAR® Serological Pipettes , Greiner Bio One International)
Mycobacterium tuberculosis
Note: Any strain or different species can be used.
Methylated spirit (Fisher Scientific, catalog number: 11492874 )
RPMI 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: A1049101 )
EDTA powder (Fisher Scientific, catalog number: 10522965 )
Versene solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15040033 )
Hank’s balanced salt solution (HBSS), no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14170112 )
HBSS, calcium, magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
Human AB serum (Sigma-Aldrich, catalog number: H4522-100ML )
Water (Millipore double distilled water, sterile)
Ultrapure alginate PRONOVATM UP MVG 10 mg (FMC, NovaMatrix, catalog number: 4200106 )
1 N NaOH cell culture grade solution (Sigma-Aldrich, catalog number: S2770-100ML )
1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630106 )
7.5% NaHCO3 solution (Thermo Fisher Scientific, catalog number: 25080060 )
Human collagen type I (3 mg/ml) (Advanced Biomatrix, catalog number: 5007-A )
0.01 N HCl (pH = 2.0)
Calcium chloride, 96%, extra pure, powder, anhydrous, ACROS OrganicsTM (Thermo Fisher Scientific, catalog number: 10021681)
Manufacturer: Acros Organics, catalog number: 349615000 .
Sodium citrate dihydrate (Fisher Scientific, catalog number: 10396430 )
3% alginate (w/v) (see Recipes)
Collagen-alginate mix (1:1 ratio) (see Recipes)
CaCl2 precipitation bath (see Recipes)
De-capsulating solution (see Recipes)
Equipment
Oven (Genlab Drying Oven, UK)
Plastic beakers (2.5 L, Thermo Fisher Scientific)
Class I/III biosafety cabinet
Borosilicate crystalizing glass beakers with spout (VWR, catalog number: 216-0068 ) (5 beakers)
Magnetic stirrers (1 cm, 5 pieces)
Sterile scissors
Thermo ScientificTM NalgeneTM Polypropylene Scissor-Type forceps (Thermo Fisher Scientific, Thermo ScientificTM)
37 °C, 5% CO2 incubator
Centrifuge (Eppendorf, model: 5427 R )
Electrostatic Bead Generator VARv1 (Nisco Engineering, model: VAR v1 )
Test-Tube-rotator (Bibby Scientific, model: STR4 )
PHD ULTRATM CP syringe pump (Harvard Apparatus, model: PHD ULTRATM CP Syringe Pump )
Jack for height adjustment (Nisco Engineering, catalog number: PE-01162 )
Procedure
SOP for bioelectrospray pre-infection
Note: This is the optimized protocol for encapsulation of peripheral blood mononuclear cells (PBMCs) after overnight infection with Mycobacterium tuberculosis. If the organism of interest is Mycobacterium tuberculosis, the reader should assume that the experiment is done under Class I/III biosafety cabinet in a standard biosafety level 3 lab under approved institutional standards of practice. If the work requires being undertaken in a standard biosafety level 2 lab, one can fumigate the whole machine according to the respective laboratory SOP before taking out from the BSL3 laboratory. The bioelectrospray technique is always performed in a Class I/III biosafety cabinet, with the doors of the bioelectrosprayer kept closed during microsphere generation as an extra level of containment. A Class II biosafety cabinet is inherently not safe to do the procedure. Once the microspheres are formed, they can be transferred for the subsequent steps either to Class II biosafety cabinet or continue in Class I/III biosafety cabinet. The protocol spans three days. Modification for other infections or other biological modelling is possible.
Day-1
Sterilize the following items in plastic beakers and dry them in an oven.
Five 150 ml Borosilicate glass beakers with spout
Magnetic stirrers (1 cm length) (5 pieces)
Female Luer Thread Style Connectors
Scissors
Forceps (different sizes)
Tissue paper
Sterile silicon tubes with connector attached at the end (3 pieces, 45 cm in length)
Prepare alginate suspension. Alginate is a natural product and there is variability on the product depending on the species of alginate and environmental conditions. In all studies our group have conducted so far, all the procedures were done with medium viscosity G dominant alginate with viscosity above 250 mPas.
Prepare 3% alginate in HBSS without Ca/Mg, with phenol red under sterile conditions and mix it with the buffers to form alginate mix.
Day-2
Prepare the alginate-collagen matrix as in Recipe 2 (Figure 1).
Figure 1. Preparation of cells for encapsulation. Cells are recovered from a 75 cm2 flask and pelleted in a Falcon by centrifugation, and then mixed with alginate-collagen matrix in a 7 ml bijou container (usually 25 million cells/5ml of matrix mix). Also see Video 1.
Video 1. Mixing alginate with PBMCs prior to bioelectrospraying
Extract PBMCs according to standard protocol for separating PBMCs from blood by density centrifugation. One can use either the buffy coat cells which are commercially available or whole blood isolated PBMCs.
Before the final wash, re-suspend the cells in 50 ml of HBSS without Ca/Mg and take 15 µl of cell suspension for cell counting (dilute 10x further if working with leukocyte cones isolated from 500 ml blood). Place the re-suspended cells in the fridge until use.
Count the cells, and calculate the total cells required for final concentration of 5 x 106 cells/ml once re-suspended in cell-alginate-mix.
Pipette off appropriate number of isolated PBMCs and then pellet by spinning at 320 x g, 8 min, 4 °C, in a 50 ml Falcon tube. Discard supernatant and add 30 ml of complete RPMI medium (ampicillin, glutamine) to the PBMC pellet and re-suspend.
Infect PBMCs with appropriate multiplicity of infection (MOI) of Mtb (our experimental standard is MOI 0.1).
Transfer the infected PBMCs into a 75 cm2 flask.
Leave overnight in a 37 °C, 5% CO2 incubator.
Day-3
Preparation of cells for encapsulation
Next day, take out the flask from the incubator; carefully transfer the contents of the flask (30 ml) to a 50 ml Falcon tube.
Add 5 ml of 5 mM EDTA (or Versene, Thermo Fisher Scientific) to the flask and incubate for 8-10 min at 37 °C, 5% CO2 incubator.
After that time, add 5 ml of HBSS without Ca, Mg (or complete RPMI medium) to the flask to dilute the effect of the detachment solution.
Scrape the bottom surface of the flask with a scraper carefully and lightly to re-suspend all remaining cells.
Transfer the 10 ml contents to the same 50 ml Falcon tube, already containing the medium (total of 40 ml). Finally rinse flask with 10 ml of HBSS without Ca, Mg (or complete RPMI medium) and add to the Falcon.
Pellet cells in the Falcon by centrifugation at 320 x g, 8 min.
Carefully, take the Falcon tube out of centrifuge and decant supernatant. Re-suspend the pelleted cells: e.g., 50 µl per 5 x 106 cells.
Prepare 5 ml alginate-collagen mix in a 7 ml bijou container. You can prepare alginate-collagen mix up to a week prior to the experiment (Video 1).
Mix well your cells with the alginate-collagen mix accordingly in a 7 ml bijou container (Usually 25 million cells/5 ml of alginate).
Keep at 4 °C on ice/in the fridge until bioelectrospraying the cell-alginate suspension.
Bioelectrospray (Figures 2, 3 and 4)
Figure 2. Nisco electrostatic encapsulator with washed and alcohol sterilized arm, sterile nozzle, sterile silicon tubes and crystalizing glass beakers. 1. Nozzle (0.7 φ) attached to the nozzle holder; 2. Electrostatic accelerator arm for the electrostatic bead generator VARv1; 3. Electrode cable; 4. Silicon tubes with connector attached at the end; 5. Borosilicate crystalizing glass beakers with spout with magnetic stirrers (1 cm); 6. Stirrer; 7. Ring on the electrostatic accelerator arm; 8. Ruler for setting correct needle height.
Figure 3. Biolelectrospraying microspheres. Matrix in syringe is injected to the bioelectrosprying machine and microspheres are formed. A syringe filled with matrix was set up on syringe pump (A) so that it will inject the matrix into silicon tube (B) connected to the electrostatic bead generator. The syringe driver is sitting on jack (C) for height adjustment. E. Unused crystalizing glass beakers on the roof of the bioelectrospray machine; F. Housing with doors to enclose the electrostatic bead generator; G. High-voltage switch on/off (white, on/green, off) which is left of potentiometers for optional peristaltic pump, agitator speed and voltage on electrode. Voltage indicator displaying 7.0 kV. Biobin (H) and old media bottle (I) containing surfanios (10%) for discarding biohazardous waste.
Figure 4. Microspheres are formed in HBSS solution with 100 mM CaCl2. A mix of collagen-alginate with cells in 2 mm diameter silicon tube at a specific rate and microspheres are formed in the gelling bath. Also see Video 3.
Items required:
Bioelectrospray sterile items (Listed above Procedure A Day-1)
Nisco Electrostatic Encapsulator with washed and alcohol sterilized arm
50 ml Falcon tube
1 M CaCl2 solution
HBSS with Ca, Mg
HBSS without Ca/Mg
Procedure:
Set the machine up at the rear of the MSC class I/III with the syringe driver on the jack adjacent to it, so that the syringe driver is equal height to the top of the bioelectrosprayer (see Videos 2 and 3).
Video 2. Setting up the bioelectrospray system
Video 3. Bioelectrospray system in operation with microspheres being formed
Adjust the syringe driver speed according to the diameter of the syringe (e.g., appropriate rate for 5 ml syringe to give 10 ml/h). The adjustment of the speed varies dependent on the syringe brand.
Prepare the Borosilicate glass beaker by placing magnetic stirrer inside.
Open the sterile silicon tubes and the nozzles and connect them to the bioelectrospray needle held in the arm of encapsulator.
Run 50 ml HBSS without Ca/Mg through the tubing using a 20 ml syringe slowly into empty Borosilicate glass beaker. This will wash the system and check the connection of the needle in the encapsulator arm.
Dry the arm with sterile tissue.
Aspirate the cell-matrix mixture into a 5 ml syringe slowly. Alginate is very viscous and so this must be performed with patience. Avoid creating bubbles. The air enclosed will be ultimately end inside the microspheres, causing them to float.
Connect the 5 ml syringe to the tube, and inject it slowly until it nears the bioelectrospray needle at the end.
Pour 100 mM CaCl2 in HBSS (without Ca/Mg) into one of the beakers until it is half-full. Put beaker under the arm of encapsulator.
Place the syringe in the syringe driver and ensure driving screw abuts end of syringe. Close the doors of the bioelectrosprayer fully.
Start bioelectrospraying by turning on the voltage and stirrer of the encapsulator, and initiating the syringe pump at 10 ml/h. Alginate-collagen mix will be ejected through the needle and the spheroids will be collected in the gelling bath. We use 7 kV voltage and 70% stirring speed. The voltage, stirring speed, affects the diameter of the microspheres and alginate type and nozzle size and further information can be found in the work of Workman and colleagues (Workman et al., 2014).
Safety Note: The electrostatic bead generator has an electric charge. Therefore, do not touch any parts when the generator is on to prevent exposure to high voltage (low current) electricity.
Once the syringe contents have been fully expelled by syringe driver, it is necessary to drive alginate mix from tubing dead space through the bioelectrosprayer needle or continue with second batch of the cell-matrix suspension.
Stop the bioelectrosprayer, replace the syringe with one containing 5 ml HBSS (without Ca/Mg), and recommence bioelectrosprayer and driver at 10 ml/h until all collagen-alginate mix is expelled and HBSS reaches the needle. This will be clear from colour and microspheres no longer form in gelling bath.
Once all alginate is bioelectrosprayed, decant microspheres to 50 ml tubes by pouring. Allow them to settle and then remove as much supernatant CaCl2 solution from the Falcon as possible to the waste bottle with a 5 ml pipette. Microspheres take about 2 min to settle and so centrifugation is not required, and may damage the spheres (Figure 5).
Add HBSS with Ca, Mg to the microspheres to total volume 50 ml. Stand Falcons in racks.
Wash the microspheres 2 x by removing HBSS with Ca, Mg with a pipette and then adding again (Video 4).
Figure 5. Microspheres in HBSS with Ca/Mg after transferred from the gelling bath. Also see Video 4.
Video 4. Decanting microspheres after generation
Aliquot microspheres to the appropriate tissue culture plate or sterile Eppendorf tubes. Aliquoting is performed using a 1 ml pipette with the end cut off with sterile scissors, to give an orifice sufficiently large to pipette up microspheres. Keep the Falcon with microspheres agitated during pipetting to keep a constant concentration within the media and avoid settling during aliquoting.
Pipette off HBSS from wells/Eppendorfs to leave microspheres only.
Add RPMI 1640 medium supplemented with 10% AB serum, and incubate at 37 °C for the duration of the experiments. Each microsphere has ~600 µm diameter and the viability of the cells after a complete procedure is 95%.
If setting up a second experimental condition (e.g., uninfected cells, different cell augmentation), then replace silicone tube and repeat steps as above. A maximum of 8 experimental conditions can be readily undertaken in one day. One should plan 1 h per 5 ml matrix, giving a total of up to 10 h for the final generation of microspheres.
De-capsulation of cells
Aspirate the microspheres into a 50 ml centrifuge tube and allow them to settle at the bottom of the tube. Then discard the supernatant carefully.
Wash the microspheres with HBSS without Ca/Mg twice. Let the microspheres settle at the bottom of the tube, then remove the supernatant.
Add 10 ml de-capsulating solution to the capsules.
Mix the suspension thoroughly prior to incubation at room temperature up to 15 min. Shake the microspheres intermittently.
When the microspheres are completely de-capsulated, their absence is visible with the naked eye. Fill the remaining tube with HBSS without Ca/Mg (or complete RPMI) at room temperature.
Centrifuge the de-capsulated cells to pellet them at 320 x g for 5 min and discard the supernatant.
Wash the cell pellet by re-suspending in HBSS followed by centrifugation.
Further use of collagenase is usually not necessary.
The de-capsulated cells can be further cultured as a monolayer or used for downstream analysis.
Data analysis
Once the microspheres are formed, one can consider them as an individual’s cells/collection of cells as the microspheres are permeable, like sponges. Microspheres can be set up in 96-well plates for assays testing viability, necrosis and apoptosis. Mycobacterial growth can be measured by luminescence if the bacterium has a luminescence reporter plasmid, or directly by counting colony forming units on Middlebrook 7H11 agar after de-capsulation. Cellular composition can be analyzed by flow cytometry after de-capsulation and paraformaldehyde fixation, and RNA analysis by de-capsulation and cellular lysis with Trizol. Data is analyzed according to standard workflows, our routine is a minimul of 2 separate donors with each experimental variable in triplicate.
Recipes
3% alginate (w/v)
Measure the alginate in aseptic conditions in BSC-II
Place approximately 1.5 g of purified sodium alginate (MVG from NovaMatrix with high glucuronic acid content ≥ 60%, viscosity > 200 mPas, and endotoxin ≤ 100 EU/g) in a sterile 50 ml tube
Weigh sterile empty Falcon tube
Weigh the Falcon tube with alginate
Determine the weight of alginate by subtracting the weight of empty Falcon tube
Add the appropriate volume of HBSS without Ca/Mg, for final percentage of 3%
Vortex the tube for approximately 3 min to partially dissolve the alginate powder then place the tube on an orbital mixer at 10 x g overnight or for two nights in a cold room
Store the alginate solution at 4 °C for short-term storage (1-2 weeks) or at -20 °C for long-term storage (one year)
Collagen-alginate mix (1:1 ratio)
Prepare the following buffers
0.05 N NaOH in 0.2 M HEPES by mixing 2.5 ml NaOH with 10 ml HEPES (stock 1 M) and adding 37.5 ml of endotoxin free water for final 50 ml solution of NaOH/HEPES solution
100 ml 7.5% NaHCO3
Human collagen (3 mg/ml dissolved aqueous solution in 0.01 N HCl [pH = 2.0])
Mix the HEPES/NaOH buffer, NaHCO3, and alginate as follows:
3% alginate: 50% of the total mix
HEPES/NaOH: 4.5% of the total mix
7.5% NaHCO3: 9% of the total mix
Filter sterilize by 0.22 µm filter (Fisher Scientific)
Add the human collagen (3 mg/ml): 36.5% of the total mix
Store the collagen-alginate solution at 4 °C for short-term storage (1-2 weeks) or at -20 °C for long-term storage (up to a year)
CaCl2 precipitation bath
Dissolve 147 g of CaCl2·2H2O and 23.8 g of HEPES in 1 L of Milli-Q H2O
Adjust the pH level to 5-6
Sterilize the solution using a 0.22 μm filter
Precipitation fluid can be stored at room temperature (6-12 months)
On the day of the experiment, mix 5 ml concentrate with 45 ml of HBSS without Ca/Mg for working solution
De-capsulating solution
Prepare and sterilize the following solutions
1 M of NaCitrate (add 294.1 g in 1 L of Milli-Q H2O)
1 M of EDTA (add 292.2 g in 1 L of Milli-Q H2O)
1 M of citric acid (add 192.1 g in 1 L of Milli-Q H2O)
Store solutions in cell culture grade plastic containers at room temperature (6-12 months)
Make 55 mM NaCitrate and 10 mM EDTA in HBSS with Ca, Mg and adjust the pH to 7.2
To prepare 100 ml of de-capsulating solution
Mix 5.5 ml of NaCitrate and 1 ml of EDTA in HBSS with Ca, Mg and adjust the pH to 7.2-7.4 and store at 4 °C for up to 2 weeks
Acknowledgments
We would like to thank S. N. Jayasinghe from University College London, United Kingdom for all the technical support and advice on the bioelectrospray technology. This work is funded by the grant from the US National Institute for Health R33AI102239, the UK National Centre for the 3Rs NC/L001039/1 and the Antimicrobial Resistance Cross Council Initiative supported by the seven research councils MR/N006631/1.
References
Al Shammari, B., Shiomi, T., Tezera, L., Bielecka, M. K., Workman, V., Sathyamoorthy, T., Mauri, F., Jayasinghe, S. N., Robertson, B. D., D'Armiento, J., Friedland, J. S. and Elkington, P. T. (2015). The extracellular matrix regulates granuloma necrosis in tuberculosis. J Infect Dis 212(3): 463-473.
Bielecka, M. K., Tezera, L. B., Zmijan, R., Drobniewski, F., Zhang, X., Jayasinghe, S. and Elkington, P. (2017). A bioengineered three-dimensional cell culture platform integrated with microfluidics to address antimicrobial resistance in tuberculosis. mBio 8:e02073-16.
Tezera, L. B., Bielecka, M. K., Chancellor, A., Reichmann, M. T., Shammari, B. A., Brace, P., Batty, A., Tocheva, A., Jogai, S., Marshall, B. G., Tebruegge, M., Jayasinghe, S. N., Mansour, S. and Elkington, P. T. (2017). Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model. eLife 6:e21283.
WHO. (2016). Global tuberculosis report 2016.
Workman, V. L., Tezera, L. B., Elkington, P. T. and Jayasinghe, S. N. (2014). Controlled generation of microspheres incorporating extracellular matrix fibrils for three-dimensional cell culture. Adv Funct Mater 24(18): 2648-2657.
Copyright: Tezera et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Tezera, L. B., Bielecka, M. K. and Elkington, P. T. (2017). Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis. Bio-protocol 7(14): e2418. DOI: 10.21769/BioProtoc.2418.
Tezera, L. B., Bielecka, M. K., Chancellor, A., Reichmann, M. T., Shammari, B. A., Brace, P., Batty, A., Tocheva, A., Jogai, S., Marshall, B. G., Tebruegge, M., Jayasinghe, S. N., Mansour, S. and Elkington, P. T. (2017). Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model. eLife 6:e21283.
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Category
Microbiology > Microbe-host interactions > Bacterium
Immunology > Immune cell function > Macrophage
Cell Biology > Cell isolation and culture > 3D cell culture
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2,419 | https://bio-protocol.org/exchange/protocoldetail?id=2419&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets
SS Stephanie Sontag
MF Malrun Förster
KS Kristin Seré
Martin Zenke
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2419 Views: 14653
Reviewed by: Xiao Li
Original Research Article:
The authors used this protocol in Apr 2017
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Original research article
The authors used this protocol in:
Apr 2017
Abstract
Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells; Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells and dendritic cells (DC) derived thereof. This makes iPS cells particularly well suited for studying molecular mechanisms of diseases, drug discovery and regenerative therapy (Grskovic et al., 2011; Bellin et al., 2012; Robinton and Daley, 2012).
DC are the major antigen presenting cells of the immune system and thus they are key players in modulating and directing immune responses (Merad et al., 2013). DC patrol peripheral and interface tissues (e.g., lung, intestine and skin) to detect invading pathogens, and upon activation they migrate to lymph nodes to activate and prime lymphocytes.
DC comprise a phenotypically heterogeneous family with functionally specialized subsets (Schlitzer and Ginhoux, 2014). Generally, classical DC (cDC) and plasmacytoid DC (pDC) are distinguished, exhibiting a classical and plasma cell-like DC morphology, respectively. cDC recognize a multitude of pathogens and secrete proinflammatory cytokines upon activation, while pDC are specialized to detect intracellular pathogens and secrete type I interferons (Merad et al., 2013; Schlitzer and Ginhoux, 2014). cDC are further divided into cross-presenting cDC1 and conventional cDC2, in the human system referred to as CD141+ Clec9a+ cDC1 and CD1c+ CD14- cDC2. Human pDC are characterized as CD303+ CD304+ (Jongbloed et al., 2010; Joffre et al., 2012; Swiecki and Colonna, 2015).
To investigate subset specification and function of human DC, we established a protocol to generate cDC1, cDC2 and pDC in vitro from human iPS cells (or ES cells) (Sontag et al., 2017). Therefore, we differentiated iPS cells (or ES cells), via mesoderm commitment and hemato-endothelial specification, into CD43+ CD31+ hematopoietic progenitors. Subsequently, those were seeded onto inactivated OP9 stromal cells with FLT3L, SCF, GM-CSF and IL-4 or FLT3L, SCF and GM-CSF to specify cDC1 and cDC2, or cDC1 and pDC, respectively.
Keywords: iPS cells ES cells Hematopoiesis Hematopoietic differentiation Human dendritic cells Dendritic cell differentiation
Background
DC and their development have mostly been studied in mice (Belz and Nutt, 2012; Merad et al., 2013; Schlitzer and Ginhoux, 2014). Owing to their rarity in non-lymphoid tissues and the restricted access to human lymphoid tissues, studying DC in humans is challenging (Jongbloed et al., 2010; Villadangos and Shortman, 2010). Yet, understanding developmental pathways, origins and mechanisms during DC subset specification is important in order to generate autologous DC subsets in vitro for therapeutic applications (e.g., anti-tumor agents). iPS cells (and ES cells) with their unlimited self-renewal and differentiation potential raise hopes that DC can be generated and modified (e.g., loaded with patient and disease specific antigens) in high numbers and quality in vitro to study DC development and function and to improve DC immunotherapies (Sontag et al., 2017).
Several groups have generated monocyte derived DC from iPS cells (and ES cells) with conventional granulocyte macrophage colony stimulating factor (GM-CSF)/interleukin 4 (IL-4) protocols, but such DC represent inflammatory DC and are not subset specific (Senju et al., 2007; Tseng et al., 2009; Choi et al., 2011; Senju et al., 2011; Belz and Nutt, 2012; Rossi et al., 2012; Yanagimachi et al., 2013; Li et al., 2014). From mouse studies, it is known that fms-like tyrosine kinase ligand (FLT3L) is the key cytokine for DC development and that the combination of FLT3L and GM-CSF signaling specifies DC subsets in vivo (Gilliet et al., 2002; Schmid et al., 2010). Recently, human cDC1, cDC2 and pDC were generated from cord blood (CB) with FLT3L, stem cell factor (SCF) and GM-CSF on MS5 stromal cells (Lee et al., 2015). Additionally, Poulin et al. (2010) reported on the generation of cDC1 from CB with FLT3L, SCF, GM-CSF and IL-4 in a feeder-free environment (Poulin et al., 2010). In contrast, Silk et al. (2012) described the differentiation of cDC1 in a feeder-free GM-CSF/IL-4 system (Silk et al., 2012). However, recent genome wide transcriptional profiling studies highlight the impact of microenvironmental cues during DC development, indicating that feeder support is important (Lundberg et al., 2013). Therefore, we used FLT3L, SCF, GM-CSF and IL-4 (referred to as FSG4) and FLT3L, SCF and GM-CSF (referred to as FSG) in combination with OP9 stromal cells to generate cDC1, cDC2 and pDC from iPS cell (or ES cell) derived hematopoietic progenitors.
Here we describe a two-step protocol: First, human iPS cells (or ES cells) are differentiated into hematopoietic progenitors (adapted from Kennedy et al., 2012). Second, these hematopoietic stem/progenitor cells are then further differentiated into cDC1, cDC2 and pDC (Sontag et al., 2017).
Materials and Reagents
6-well tissue culture plates (TPP Techno Plastic Products, catalog number: 92006 )
10 cm microbiology grade Petri dishes (SARSTEDT, catalog number: 82.1473.001 )
50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
70 µm cell strainer (Greiner Bio One International, catalog number: 542070 )
40 µm cell strainer (Greiner Bio One International, catalog number: 542040 )
Top bottle filter (TPP Techno Plastic Products, catalog number: 99505 )
5 ml Serological pipettes (Corning, Falcon®, catalog number: 357543 )
10 ml Serological pipettes (Corning, Falcon®, catalog number: 357551 )
25 ml Serological pipettes (Corning, Falcon®, catalog number: 357525 )
Pipette tips 10 µl (STARLAB INTERNATIONAL, catalog number: S1180-3810 )
Pipette tips 20 µl (STARLAB INTERNATIONAL, catalog number: S1120-1810 )
Pipette tips 200 µl (STARLAB INTERNATIONAL, catalog number: S1120-8810 )
Pipette tips 1,000 µl (STARLAB INTERNATIONAL, catalog number: S1126-7810 )
Collagenase IV (Thermo Fisher Scientific, GibcoTM, catalog number: 17104019 )
Knockout-Dulbecco’s modified Eagle medium (KO-DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 10829018 )
Gelatin (Sigma-Aldrich, catalog number: G1890 )
1-Thiogylcerol (Sigma-Aldrich, catalog number: M1753 )
L-ascorbic acid (Sigma-Aldrich, catalog number: A4403 )
Bovine serum albumin (BSA) low endotoxin (PAA, catalog number: K31-011 )
1x Dulbecco’s phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190094 )
HumanKine bone morphogenic protein 4 (BMP4) research grade (Miltenyi Biotec, catalog number: 130-110-921 )
Recombinant human basic fibroblast growth factor (bFGF) (PeproTech, catalog number: 100-18B )
Recombinant human FLT3L (PeproTech, catalog number: 300-19 )
Recombinant human GM-CSF (PeproTech, catalog number: 300-03 )
Recombinant human insulin-like growth factor 1 (IGF1) (PeproTech, catalog number: 100-11 )
Human interleukin 3 (IL-3) research grade (Miltenyi Biotec, catalog number: 130-094-193 )
Human IL-4 research grade (Miltenyi Biotec, catalog number: 130-095-373 )
Human SCF research grade (Miltenyi Biotec, catalog number: 130-096-693 )
Human thrombopoietin (TPO) research grade (Miltenyi Biotec, catalog number: 130-094-013 )
Recombinant human vascular endothelial growth factor (VEGF) (PeproTech, catalog number: 100-20 )
1x StemPro-34 SFM (Thermo Fisher Scientific, catalog number: 10639011 )
Penicillin-streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
L-glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
Hyper-interleukin 6 (IL-6) (kindly provided by S. Rose John, Institute of Biochemistry, Medical Faculty, Christian-Albrechts-University, Kiel, Germany, [Fischer et al., 1997], see also Notes)
α-Minimal essential medium (α-MEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 12571063 )
β-mercaptoethanol (50 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
Collagenase IV solution (1 mg/ml) (see Recipes)
0.1% gelatin solution (see Recipes)
1-Thioglycerol stock solution (100 mM) (see Recipes)
L-ascorbic acid stock solution (50 mg/ml) (see Recipes)
0.1% BSA solution (see Recipes)
BMP4 stock solution (25 µg/ml) (see Recipes)
bFGF stock solution (100 µg/ml) (see Recipes)
FLT3L stock solution (25 µg/ml) (see Recipes)
GM-CSF stock solution (100 µg/ml) (see Recipes)
IGF1 stock solution (40 µg/ml) (see Recipes)
IL-3 stock solution (150 µg/ml) (see Recipes)
IL-4 stock solution (20 µg/ml) (see Recipes)
SCF stock solution (100 µg/ml) (see Recipes)
TPO stock solution (20 µg/ml) (see Recipes)
VEGF stock solution (100 µg/ml) (see Recipes)
Hematopoietic progenitor basal differentiation medium (see Recipes)
Induction medium d0 (see Recipes)
Induction medium d1 (see Recipes)
Induction medium d2 (see Recipes)
Induction medium d4 (see Recipes)
Induction medium d6 (see Recipes)
OP9 culture medium (see Recipes)
DC differentiation basal medium (see Recipes)
DC differentiation FSG4 medium (see Recipes)
DC differentiation FSG medium (see Recipes)
Equipment
1,000 µl pipette (Gilson, catalog number: F123602 )
200 µl pipette (Gilson, catalog number: F123601 )
20 µl pipette (Gilson, catalog number: F123600 )
10 µl pipette (Gilson, catalog number: F144802 )
Pipetboy (INTEGRA Biosciences, catalog number: 155000 )
Water bath (JULABO, model: SW22 )
Inverted light microscope (Leica Microsystems, model: Leica DM IL LED )
Autoclave
Centrifuge (Thermo Fisher Scientific, model: HeraeusTM Multifuge 3 L )
Flow hood (Heraeus)
Automatic CO2 incubator with nitrogen supply and O2 sensor (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM 240i , catalog number: 51026331)
Vacuum pump (INTEGRA Biosciences, catalog number: 158300 )
Standard fridge
Standard non-defrosting freezer
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sontag, S., Förster, M., Seré, K. and Zenke, M. (2017). Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets. Bio-protocol 7(15): e2419. DOI: 10.21769/BioProtoc.2419.
Download Citation in RIS Format
Category
Stem Cell > Pluripotent stem cell > Cell induction
Cell Biology > Cell isolation and culture > Cell differentiation
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242 | https://bio-protocol.org/exchange/protocoldetail?id=242&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
5-Hydroxymethylcytosine (5-hmC) Specific Enrichment
KS Keith E Szulwach
CS Chun-Xiao Song
CH Chuan He
PJ Peng Jin
Published: Vol 2, Iss 15, Aug 5, 2012
DOI: 10.21769/BioProtoc.242 Views: 10282
Original Research Article:
The authors used this protocol in Jan 2011
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Original research article
The authors used this protocol in:
Jan 2011
Abstract
5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA modification in mammalian genomes. This protocol is to be a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically, which could in turn be used for high-throughput mapping via next-generation sequencing.
Keywords: 5-hydroxymethylcytocine Capture Sequencing
Materials and Reagents
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
Trizma base (Tris) (Sigma-Aldrich, catalog number: T1503 )
HEPES (GenScript USA, catalog number: C01621 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128 )
Tween-20 (Sigma-Aldrich, catalog number: P9416 )
DBCO-S-S-PEG3-Biotin conjugate (Click Chemistry Tools, catalog number: A112P3 )
1,4-Dithiothreitol, ultrapure (DTT) (Roche Applied Science, catalog number: 3117006001 )
Hydroxymethyl Collector (Active Motif, catalog number: 55013 )
Wizard Genomic DNA Purification Kit (Promega Corporation, catalog number: A1120 )
10 kDa Amicon Ultra-0.5 ml Centrifugal Filters (EMD Millipore, catalog number: UFC501008 )
QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28304 )
Micro Bio-Spin 6 Column (Bio-Rad Laboratories, catalog number: 732-6222 )
Dynabeads MyOne Streptavidin C1 (Life Technologies, Invitrogen™, catalog number: 650-01 )
Qiagen MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
UltraPure Agarose (Life Technologies, Invitrogen™, catalog number: 16500500 )
β-glucosyltransferase (β-GT) (New England Biolabs)
10x β-GT reaction buffer (see Recipes)
2x binding and washing (B&W) buffer (see Recipes)
TE (see Recipes)
Equipment
Sonication device (Covaris)
Desktop centrifuge
Water bath (Thermo Fisher Scientific)
Gel running apparatus (Bio-Rad Corporation)
Nanodrop 1000 (Thermo Fisher Scientific)
Labquake tube shaker (Barnstead/Thermolyne)
Magnetic separation stand (Promega Corporation, catalog number: Z5342 )
Qubit 2.0 Fluorometer (Life Technologies, Invitrogen™)
Procedure
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Category
Molecular Biology > DNA > DNA modification
Biochemistry > Carbohydrate > Glucose
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2,420 | https://bio-protocol.org/exchange/protocoldetail?id=2420&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS
MB Marina Borisova
CM Christoph Mayer
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2420 Views: 8316
Reviewed by: Masahiro MoritaKanika Gera
Original Research Article:
The authors used this protocol in Oct 2016
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Oct 2016
Abstract
We describe here in detail a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based method to determine N-acetylmuramic acid-6-phosphate (MurNAc-6P) in bacterial cell extracts. The method can be applied to both Gram-negative and Gram-positive bacteria, and as an example we use Escherichia coli cells in this study. Wild type and mutant cells are grown for a defined time in a medium of choice and harvested by centrifugation. Then the cells are disintegrated and soluble cell extracts are generated. After removal of proteins by precipitation with acetone, the extracts are analyzed by HPLC-MS. Base peak chromatograms of wild type and mutant cell extracts are used to determine a differential ion spectrum that reveals differences in the MurNAc-6P content of the two samples. Determination of peak areas of extracted chromatograms of MurNAc-6P ((M-H)- = 372.070 m/z in negative ion mode) allows quantifying MurNAc-6P levels, that are used to calculate recycling rates of the MurNAc-content of peptidoglycan.
Keywords: Bacteria Cell wall metabolism Peptidoglycan recycling Cytosolic metabolites LC-MS Base peak chromatogram (BPC) Extracted ion chromatogram (EIC) MurNAc-6P accumulation
Background
Large parts of the peptidoglycan cell wall of bacteria are steadily turned over and possibly recovered (recycled) during bacterial growth. A key compound of the peptidoglycan recycling metabolism is N-acetylmuramic acid-6-phosphate (MurNAc-6P), which accumulates in a MurNAc-6P etherase (MurQ) mutant of Escherichia coli (Jaeger et al., 2005; Uehara et al., 2006). MurQ orthologs are found in many bacteria, including Gram-positive bacteria (Litzinger et al., 2010; Reith and Mayer, 2011). MurNAc-6P accumulation in murQ mutants recently proved recycling of the MurNAc-content of the bacterial cell wall in Gram-positive bacteria and was used to quantify intracellular MurNAc-6P levels, which allowed determining peptidoglycan recycling rates (Borisova et al., 2016).
Materials and Reagents
50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
Micro-tubes 2 ml (SARSTEDT, catalog number: 72.691 )
Micro-tubes 2 ml with cap (SARSTEDT, catalog number: 72.694 )
Glass beads (0.25 to 0.5 mm) (Carl Roth, catalog number: A553.1 )
Escherichia coli strains: MC4100 (wild type) and TJ2e (∆murQ) (Jaeger et al., 2005)
Acetone (CH3COCH3) (Sigma-Aldrich, catalog number: 34850-2.5L )
Ammonium formate (NH4HCOO) (VWR, catalog number: 17843-50G )
Acetonitrile (CH3CN) (Avantor Performance Materials, J.T. Baker®, catalog number: 9012-03 )
Millipore ultrapure water (autoclaved)
BactoTM yeast extract (BD, BactoTM, catalog number: 212720 )
BactoTM tryptone (BD, BactoTM, catalog number: 211699 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-5KG-R )
Propan-2-ol (Sigma-Aldrich, catalog number: 34863-2.5L-M )
Formic acid (HCOOH) (VWR, catalog number: 56302-50ML )
Sodium hydroxide (NaOH), 1 N (VWR, catalog number: 31627.290 )
Medium Luria Bertani (LB) broth (see Recipes)
LC-MS calibrant (10 mM sodium formate) (see Recipes)
HPLC buffer A (see Recipes)
Equipment
1,000 ml Erlenmeyer flasks with chicane
100 ml Erlenmeyer flasks with chicane
Shaker (set at 160 rpm) (Eppendorf, New BrunswichTM, model: Excella® E10 )
Pipette controller (BrandTech Scientific, model: accu-jet® pro )
Microcentrifuge (Thermo Fischer Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 )
Medium bench centrifuge (Thermo Fischer Scientific, model: HeraeusTM Biofuge Pico )
Cell density meter (Biochrom, model: Biochrom WPA CO8000 )
Cell disrupter (GMI, model: Thermo Savant FastPrep 120 )
Rotational vacuum concentrator (Martin Christ Gefriertrocknungsanlagen, model: RVC 2-18 CDplus )
Gemini® 5 µm 110Å, 150 x 4.6 mm LC column (Phenomenex, catalog number: 00F-4435-E0 )
Mass spectrometer (Bruker, model: micrOTOF focus II )
High-performance liquid chromatography (Thermo Fischer Scientific, Thermo ScientificTM, model: UltimateTM 3000 RS )
Software
Chromeleon Xpress (Dionex)
MicroTOF control Version 3.0 (Bruker Daltonics)
Bruker Compass HyStar Version 3.2 (Bruker Daltonics)
Compass Data Analysis Version 4.0 (Bruker Daltonics)
MetaboliteDetect 2.0 (Bruker Daltonics)
GraphPad Prism 6 (San Diego, CA, USA)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Borisova, M. and Mayer, C. (2017). Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS. Bio-protocol 7(15): e2420. DOI: 10.21769/BioProtoc.2420.
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Category
Microbiology > Microbial physiology > Membrane property
Microbiology > Microbial biochemistry > Other compound
Biochemistry > Carbohydrate > Peptidoglycan
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2,421 | https://bio-protocol.org/exchange/protocoldetail?id=2421&type=0 | # Bio-Protocol Content
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Peer-reviewed
Polyamine and Paraquat Transport Assays in Arabidopsis Seedling and Callus
HC Haoxi Chai
YS Yun Shen
Huazhong Shi
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2421 Views: 6751
Edited by: Marisa Rosa
Reviewed by: Joëlle Schlapfer
Original Research Article:
The authors used this protocol in Dec 2016
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Abstract
Polyamines (PAs) are polycationic compounds found in all living organisms and play crucial roles in growth and survival. We here show the ‘Polyamine and paraquat (PQ) transport assay’ protocol, which can be used to examine the uptake activity of PA/PQ transporters. We have used this protocol to demonstrate that PUT3 in Arabidopsis is a polyamine transporter and is able to take up spermidine and its analog paraquat.
Keywords: Arabidopsis thaliana Polyamine Paraquat Transport Uptake
Background
PAs are involved in gene regulation by interacting with and modulating the functions of anionic macromolecules such as DNA, RNA and proteins. In living cells, PAs’ contents must be regulated to maintain the cellular hemostasis. In higher plants, three major polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm), are present in either free form or conjugated forms with other molecules (Gill and Tuteja, 2010). In yeast, four plasma membrane polyamine transporters, DUR3, SAM3, GAP1 and AGP2 were identified (Uemura et al., 2007). In Arabidopsis, five putative polyamine uptake transporters (PUT1-PUT5) were identified and PUT1-3 have been experimentally validated as polyamine transporters (Mulangi et al., 2012; Li et al., 2013). Our protocol described below has successfully confirmed that PUT3 is an influx transporter for polyamines and paraquat, and PQ/Spd uptake is impaired in the put3 mutant (Shen et al., 2016).
Materials and Reagents
Pipette tips
Plastic Petri dish (VWR, catalog number: 25384-326 )
Parafilm
1.5 ml Eppendorf centrifuge tube (VWR, catalog number: 20170-355 )
Filter paper
Blue pestle (DWK Life Sciences, Kimble, catalog number: 749521-1500 )
Cuvette (VWR, catalog number: 414004-051 )
Syringe filters, 0.2 µm pore size (VWR, catalog number: 28145-475 )
Syringe
Arabidopsis thaliana ecotype Columbia (Col-0) and mutant line lhr1 (put3)
ScintiVerseTM BD Cocktail (Fisher Scientific, catalog number: SX18-4 )
Clorox Bleach
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
(2,4-Dichlorophenoxy) acetic acid sodium salt monohydrate (Sigma-Aldrich, catalog number: D6679 )
Kinetin (Duchefa Biochemie, catalog number: K0905 )
Sodium hydroxide (NaOH)
Murashige & Skoog Basal Salt Mixture (PhytoTechnology Laboratories, catalog number: M524 )
Sucrose (Sigma-Aldrich, catalog number: S0389 )
Agar (Sigma-Aldrich, catalog number: A1296 )
Low melting point agarose (Sigma-Aldrich, catalog number: A9414 )
Methyl viologen dichloride hydrate (Sigma-Aldrich, catalog number: 856177 )
Spermidine (MP Biomedicals, catalog number: 02152068 )
Paraquat-methyl-14C dichloride hydrate (Sigma-Aldrich, catalog number: 313947 )
Note: This product has been discontinued.
Spermidine trihydrochloride [Terminal Methylenes-3H (N)] (PerkinElmer, catalog number: NET522001MC )
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 )
2-Mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
Note: This product has been discontinued.
Seed sterilization solution (see Recipes)
2,000x 2,4-D (9.05 mM) (see Recipes)
2,000x kinetin (1.86 mM) (see Recipes)
Callus induction solid medium (see Recipes)
Callus induction liquid medium (see Recipes)
½ MS solid medium (see Recipes)
½ MS liquid medium (see Recipes)
200 µM non-14C-labeled PQ solution (see Recipes)
2.04 mM non-3H-labeled Spd solution (see Recipes)
40.37 µM non-3H-labeled Spd solution (see Recipes)
Solution 1: 14C-labeled PQ solution (see Recipes)
Solution 2: 14C-labeled PQ solution (see Recipes)
Solution 3: 3H-labeled Spd solution (see Recipes)
Solution 4: 3H-labeled Spd solution (see Recipes)
Solution 5: 3H-labeled Spd solution (see Recipes)
Solution 6: 3H-labeled Spd solution (see Recipes)
1 M Tris-HCl, pH 7.5 (see Recipes)
0.5 M EDTA, pH 8.0 (see Recipes)
Crude protein extraction buffer (see Recipes)
Equipment
Pipettes
pH meter
Weighing balance
Laminar flow hood
Stirring bar
Magnetic stirrer (VWR, model: 200 Mini-stirrer )
Vortex (Fisher Scientific, model: Vortex-Genie 2 )
Scintillation counter (Beckman Coulter, model: LS-6500 )
Centrifuge (Beckman Coulter, model: Microfuge® 22R , catalog number: 368831)
Spectrophotometer (Bio-Rad Laboratories, model: SmartSpec Plus, catalog number: 1702525 )
Note: This product has been discontinued.
Autoclave
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chai, H., Shen, Y. and Shi, H. (2017). Polyamine and Paraquat Transport Assays in Arabidopsis Seedling and Callus. Bio-protocol 7(15): e2421. DOI: 10.21769/BioProtoc.2421.
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Category
Plant Science > Plant physiology > Tissue analysis
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2,422 | https://bio-protocol.org/exchange/protocoldetail?id=2422&type=0 | # Bio-Protocol Content
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Peer-reviewed
Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords
Oliver Wirths
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2422 Views: 27298
Reviewed by: Fanny Ehret
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western blotting.
Keywords: Protein extraction Homogenization Soluble fraction SDS Brain
Background
This simple and reproducible protocol of brain tissue protein fractionation details the initial separation of a total protein homogenate into a soluble and an insoluble fraction. It can also be applied also to other tissue samples and yields a soluble fraction containing hydrophilic proteins and an insoluble fraction consisting of more hydrophobic proteins. Following an initial homogenization in a lysis buffer containing no detergent, the supernatant including the soluble protein fraction is removed and the pellet containing the insoluble fraction can be further extracted using Sodium Dodecyl Sulfate (SDS) as detergent to ensure entire cell lysis (see Figure 1). This approach can facilitate the analysis of low-abundance proteins by reducing the complexity of the sample.
Figure 1. Flow-chart describing the sequential extraction procedure
Materials and Reagents
Pipette tips
10 µl pipette tips (SARSTEDT, catalog number: 70.1130 )
200 µl pipette tips (SARSTEDT, catalog number: 70.760.002 )
1,000 µl pipette tips (SARSTEDT, catalog number: 70.762 )
Reaction tubes
1.5 ml tubes (SARSTEDT, catalog number: 72.706 )
2 ml tubes (SARSTEDT, catalog number: 72.691 )
Mice (protocol has been tested with male and female C57Bl/6 mice of 8-52 weeks)
Sodium chloride (NaCl) (≥ 99.5%) (Carl Roth, catalog number: 3957 )
Tris (≥ 99.9%, p.a.) (Carl Roth, catalog number: 4855 )
Protease inhibitor cocktail (cOmpleteTM Mini EDTA-free EasyPack) (Roche Diagnostics, catalog number: 04693159001 )
Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
Sodium dodecyl sulfate (SDS) (≥ 99.5%) (Carl Roth, catalog number: 2326 )
Benzonase (Sigma-Aldrich, catalog number: E1014 )
Lysis buffer (see Recipes)
2% SDS (see Recipes)
Equipment
Pipettes (Research 0.5-10 µl, 10-100 µl, 100-1,000 µl) (Eppendorf)
Glass homogenizer (2 ml) (Carl Roth, catalog number: TT57.1 )
Teflon pestle (Carl Roth, catalog number: TT63.1 )
Stirring device (Ingenieurbüro CAT M. Zipperer, model: R 50D )
Heraeus Biofuge Stratos (Rotor) (Heraeus Holding, catalog number: 3332 )
Ultrasound sonicator (Emerson Electric, BRANSON, model: 150 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wirths, O. (2017). Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords. Bio-protocol 7(15): e2422. DOI: 10.21769/BioProtoc.2422.
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Category
Neuroscience > Cellular mechanisms > Protein isolation
Molecular Biology > Protein > Isolation
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Could you please expand on some resources for processing pellet 2 in step B8 of this procedure? Are they any alternatives to formic acid to look into?
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Dec 29, 2022
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2,423 | https://bio-protocol.org/exchange/protocoldetail?id=2423&type=0 | # Bio-Protocol Content
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Peer-reviewed
Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords
Oliver Wirths
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2423 Views: 13851
Reviewed by: Fanny Ehret
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like Western-blot or ELISA in e.g., mouse models of Alzheimer’s disease or other neurodegenerative conditions.
Keywords: Brain Synaptosome Fractionation Homogenization Extraction Synapse
Background
Analyzing synaptosomes, representing isolated synaptic terminals from neurons, can yield valuable information on synaptic integrity in diverse neurological diseases. They contain membrane-bound compartments that detach from axon terminals after brain homogenization under certain conditions. The current protocol describes a fast and easy method for the enrichment of crude synaptosomal fractions (see Figure 1). These can be either used for quantification of synaptic proteins by Western-blot or can be further purified using density gradient centrifugation to yield highly purified synaptosome subfractions (Gurd et al., 1974). The preparation of crude synaptosomal fractions might be sufficient to assess e.g., the amount of pre- and post-synaptic proteins like SNAP25 or post-synaptic density protein 95 (PSD95) in e.g., mouse models with a neurodegenerative phenotype (Breyhan et al., 2009; Saul and Wirths, 2017).
Figure 1. Flow-chart describing the sequential extraction procedure
Materials and Reagents
Pipette tips
10 µl pipette tips (SARSTEDT, catalog number: 70.1130 )
200 µl pipette tips (SARSTEDT, catalog number: 70.760.002 )
1,000 µl pipette tips (SARSTEDT, catalog number: 70.762 )
Reaction tubes
1.5 ml tubes (SARSTEDT, catalog number: 72.706 )
2 ml tubes (SARSTEDT, catalog number: 72.691 )
Mice (Protocol has been tested with male and female C57Bl/6 mice of 8-52 weeks)
Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S0389 )
HEPES (≥ 99.5%) (Sigma-Aldrich, catalog number: H3375 )
Protease inhibitor cocktail (cOmpleteTM Mini EDTA-free EasyPack) (Roche Diagnostics, catalog number: 04693159001 )
Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
Sodium chloride (NaCl) (≥ 99.5%) (Carl Roth, catalog number: 3957 )
Potassium chloride (KCl) (≥ 99%) (Carl Roth, catalog number: P017 )
Di-Sodium hydrogen phosphate (Na2HPO4) (≥ 98%) (Carl Roth, catalog number: P030 )
Potassium di-hydrogen phosphate (KH2PO4) (≥ 99%) (Carl Roth, catalog number: 3904 )
Lysis buffer (pH 7.5) (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)
Equipment
Pipettes (Research 0.5-10 µl, 10-100 µl, 100-1,000 µl) (Eppendorf)
Heraeus Biofuge Stratos (Rotor) (Heraeus Holding, catalog number: 3332 )
Ultrasound sonicator (Emerson Electric, BRANSON, model: 150 )
Stirring device (Ingenieurbüro CAT M. Zipperer, model: R 50D )
Glass homogenizer (2 ml) (Carl Roth, catalog number: TT57.1 )
Teflon pestle (Carl Roth, catalog number: TT63.1 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wirths, O. (2017). Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords. Bio-protocol 7(15): e2423. DOI: 10.21769/BioProtoc.2423.
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Category
Neuroscience > Cellular mechanisms > Synaptic physiology
Molecular Biology > Protein > Expression
Molecular Biology > Protein > Protein-protein interaction
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2,424 | https://bio-protocol.org/exchange/protocoldetail?id=2424&type=0 | # Bio-Protocol Content
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Peer-reviewed
Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants
AV Alexandra S. Vagasi
MR Mohammad M. Rahman
SC Snehal N. Chaudhari
EK Edward T. Kipreos
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2424 Views: 8310
Edited by: Neelanjan Bose
Original Research Article:
The authors used this protocol in Jul 2016
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Original research article
The authors used this protocol in:
Jul 2016
Abstract
The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016.
Keywords: C. elegans Primary culture Germ stem cells Tissue culture
Background
C. elegans hermaphrodite germ cells are generated in two adult stem cell niches located in the distal regions of the two gonad arms (Hansen and Schedl, 2013; Kimble and Seidel, 2013). In wild-type hermaphrodites, mitotic germ cells are restricted to the distal, stem cell niche regions of the gonad arms. Wild-type germ cells are syncytial, and contain an opening to a common cytoplasm that extends through the central region of the gonad arms. C. elegans tumorous germline mutants have increased mitotic proliferation of germ cells throughout the gonad. We discovered that tumorous germline mutants generally have fully cellularized germ cells that contain intact plasma membranes (Chaudhari et al., 2016). This cellularization allows the isolation of the germ cells and their maintenance in culture. This protocol describes the methodology and tissue culture medium to isolate and maintain germ cells from tumorous mutants in culture. While a culture medium has been described for the primary culture of C. elegans embryonic and larval cells (Strange et al., 2007; Zhang and Kuhn, 2013), germ cells do not survive in this medium (Chaudhari et al., 2016). We created a culture medium for germ cells that is called CeM1 for ‘C. elegans medium 1’. We anticipate that other iterations of the medium could be given subsequent numbers, e.g., ‘CeM2’. This protocol, first reported in Chaudhari et al., 2016, allows the isolation of essentially pure populations of germ cells and their maintenance in in vitro primary cultures. This culture system can allow new experimental approaches to probe germ cell biology in C. elegans.
Materials and Reagents
Polystyrene tubes, 15 ml (Corning, Falcon®, catalog number: 352099 )
Polypropylene tubes, 15 ml (Corning, catalog number: 25319-15 )
Polypropylene tubes, 50 ml (Corning, catalog number: 25330-50 )
Filter pipette tips, 100-1,000 µl (Corning, catalog number: 4809 )
Filter pipette tips, 20-200 µl (Fisher Scientific, catalog number: 02-707-430 )
Filter pipette tips, 2-20 µl (Fisher Scientific, catalog number: 02-707-435 )
Filter pipette tips, 0.1-10 µl (Fisher Scientific, catalog number: 02-707-439 )
Filter units 150 ml, PES 0.22 µm (EMD Millipore, catalog number: SCGPU01RE )
Filter units 500 ml, PES 0.22 µm (EMD Millipore, catalog number: SCGPU05RE )
Aluminum foil
Tissue culture dish, 35 x 10 mm (Corning, Falcon®, catalog number: 353001 )
Tissue culture dish, 24-well (Corning, catalog number: 3524 )
Tissue culture dish, 96-well (Corning, catalog number: 3596 )
Cell scrapers, 39 cm, disposable (SARSTEDT, catalog number: 83.1831 )
Serological pipets, disposable 10 ml (Fisher Scientific, catalog number: 13-678-11E )
Tissue culture flask, 12.5 cm2 (Corning, Falcon®, catalog number: 353018 )
Platinum wire, 0.25 mm (Alfa Aesar, catalog number: 10288 )
Needles, 21 G x 1 ½ (BD, catalog number: 305167 )
Parafilm
0.5 ml centrifuge tube
Microcentrifuge tubes, 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
Razor blades, single edge
Paper towel
C. elegans tumorous germline mutant strain ET507, daf-16(mu86) I; cki-2(ok2105) II; glp-1(ar202) III
Escherichia coli strain OP50
Notes:
We did not perform tests to determine how materials and reagents from other manufacturers function in germ cell isolation and culture, with the exception of the use of STARSTEDT 96-well plates (non-tissue culture-treated) (STARSTEDT, catalog number: 82.1581.001 ) for the in vitro culture of germ cells, which resulted in the premature death of the germ cells.
C. elegans germline tumor mutant strain ET507 (as well as other tumorous mutant strains) and E. coli bacteria OP50 are available from the Caenorhabditis Genetics Center (CGC), http://www.cgc.cbs.umn.edu.
Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
Amberlite IRA 400-CL (Sigma-Aldrich, catalog number: 247669 )
Charcoal-dextran (Sigma-Aldrich, catalog number: C6241 )
Liquid nitrogen
Phosphate buffered saline (PBS) (GE Healthcare, HyCloneTM, catalog number: SH30256.01 )
Schneider’s insect medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720024 )
Leibovitz’s L-15 medium without phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 21083027 )
Penicillin/streptomycin (Sigma-Aldrich, catalog number: P4333 )
Hemin chloride (MP Biomedicals, catalog number: 0219402501 )
RPMI Vitamins (Sigma-Aldrich, catalog number: R7256 )
L-glutathione, reduced (Sigma-Aldrich, catalog number: G4251 )
Normocin (InvivoGen, catalog number: ant-nr-1 )
Trehalose (Sigma-Aldrich, catalog number: T0167 )
Osmolality standard, 100 mmol/kg (Wescor, catalog number: OA-010 )
Osmolality standard, 290 mmol/kg (Wescor, catalog number: OA-029 )
Osmolality standard, 1,000 mmol/kg (Wescor, catalog number: OA-100 )
Water, molecular biology grade (GE Healthcare, HyCloneTM, catalog number: SH30538.02 )
Tryptone (Fisher Scientific, catalog number: BP1421-500 )
Yeast extract (Fisher Scientific, catalog number: BP9727-2 )
Sodium chloride (NaCl) (Avantor Performance Materials, J.T. Baker®, catalog number: 3624-05 )
Sodium hydroxide (NaOH) (Avantor Performance Materials, J.T. Baker®, catalog number: 3728-01 )
Bacto-peptone (BD, BactoTM, catalog number: 211677 )
Agar (RPI, catalog number: A20020-5000 )
Cholesterol (Avantor Performance Materials, J.T. Baker®, catalog number: 1580-01 )
Ethanol, 100% (used to make 70% with distilled water) (Decon Labs, catalog number: 2716 )
Magnesium sulfate, anhydrous (MgSO4) (Avantor Performance Materials, J.T. Baker®, catalog number: 2506-01 )
Calcium chloride (CaCl2) (Avantor Performance Materials, J.T. Baker®, catalog number: 1313-01 )
Potassium phosphate, monobasic (KH2PO4) (Avantor Performance Materials, J.T. Baker®, catalog number: 3246-05 )
Sodium phosphate, heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: S373-3 )
Sodium hypochlorite, 6% (RICCA Chemical, catalog number: 7495.7-32 )
Tetracycline (Sigma-Aldrich, catalog number: 87128 )
Chloramphenicol (RPI, catalog number: C61000-25.0 )
Kanamycin (Fisher Scientific, catalog number: BP906-5 )
Ethidium homodimer (Biotium, catalog number: 40010 )
Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: D128-500 )
Hoechst 33342 (Sigma-Aldrich, catalog number: B2261 )
Calcein-AM (Biotium, catalog number: 80011 )
2xYT bacterial medium (see Recipes)
LB bacterial medium (see Recipes)
3x NGM agar plates seeded with OP50 bacteria (see Recipes)
M9 buffer (see Recipes)
Sodium hypochlorite solution (see Recipes)
Platinum-wire worm pick (see Recipes)
Tetracycline stock (see Recipes)
Chloramphenicol stock (see Recipes)
Kanamycin stock (see Recipes)
Cholesterol stock (see Recipes)
Antibiotic-enriched PBS with heat-killed bacteria (see Recipes)
Hemin chloride stock (see Recipes)
Stock of Hoechst 33342, calcein-AM, and ethidium homodimer (see Recipes)
Equipment
Temperature-controlled water bath
Table-top centrifuge with swinging bucket rotor for 15 ml and 50 ml tubes
Rotator, single speed (Barnstead Thermolyne, catalog number: C415110 )
500 ml plastic bottle
Freezing point osmometer (Advanced Digimatic Osmometer 3DII, Advanced Instruments, model: Model 3D2 )
Low-temperature incubator
Bottom illuminated stereomicroscope with frosted glass stage or frosted mirror (various Nikon or Leica models)
Airtight containers (LockandLock, various size Lock & Lock containers available from Amazon)
Phase-contrast inverted compound microscope (various Nikon, Leica, or Olympus models with 10x and 20x objectives)
Nutator (BD, catalog number: 421105 )
Hemacytometer (Reichert Bright-Line, Hauser Scientific, catalog number: 1492 )
Upright fluorescence compound microscope (various Nikon, Leica, or Olympus models with 5x, 20x, 40x, and 64x objectives)
2 L Erlenmeyer flasks (Corning, PYREX®, catalog number: 4980-2L )
Autoclave
50 ml Erlenmeyer flasks (Corning, PYREX®, catalog number: 4980-50 )
Plastic 1 L measuring cup with handle (Fisher Scientific, catalog number: 02-543-36C )
Stir bar
Bottles, 125 ml, glass (WHEATON, catalog number: 219815 )
Bunsen burner
Analytical balance
pH meter
Pipette controller (Pipet-Aid) (Drummond Scientific, catalog number: 4-000-110 )
Pasteur pipets, 9 inch, glass (Fisher Scientific, catalog number: 22-063172 )
Bulbs for Pasteur pipets (Fisher Scientific, catalog number: 03-448-21 )
Laminar-flow tissue culture hood
Aspirator
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Vagasi, A. S., Rahman, M. M., Chaudhari, S. N. and Kipreos, E. T. (2017). Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants. Bio-protocol 7(15): e2424. DOI: 10.21769/BioProtoc.2424.
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Category
Stem Cell > Germ cell > In vitro culture
Cell Biology > Cell isolation and culture > Cell isolation
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2,425 | https://bio-protocol.org/exchange/protocoldetail?id=2425&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
Xiaoou Jiang
Volker Patzel
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2425 Views: 9042
Edited by: Longping Victor Tse
Reviewed by: Kanika Gera
Original Research Article:
The authors used this protocol in Sep 2016
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Original research article
The authors used this protocol in:
Sep 2016
Abstract
Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA nuclear import signals such as SV40 enhancer sequences are molecular features that have found multiple applications in plasmid vectors to improve transgene expression. In dumbbells however, effects triggered by introns were not investigated and DNA-based nuclear import sequences have not found applications yet, presumably because dumbbell vectors have continuously been minimized with regard to size. We investigated the effects of an intron and/or SV40 enhancer derived sequences on dumbbell vector driven reporter gene expression. The implementation of a spliceable intron was found to enhance gene expression unconditionally in all investigated cell lines. Conversely, the use of the SV40 enhancer improved gene expression in a cell type-dependent manner. Though both features significantly enlarge dumbbell vector size, neither the intron nor the enhancer or a combination of both revealed a negative effect on gene expression. On the contrary, both features together improved dumbbell-driven gene expression up to 160- or 56-fold compared with plasmids or control dumbbells. Thus, it is highly recommended to consider an intron and the SV40 enhancer for dumbbell vector design. Such an advanced design can facilitate pre-clinical and clinical applications of dumbbell-shaped DNA vectors.
Keywords: Dumbbell vector Minimal DNA vector Transgene expression Genetic therapy Intron SV40 enhancer
Background
Although many genes have been expressed using dumbbell-shaped DNA vectors, most of these applications used the basic design comprising a promoter, the coding sequence (CDS), and a transcriptional terminator. Some vectors included a chimeric intron, however, it was not reported whether transgene expression was enhanced by this design (Schirmbeck et al., 2001). Here we studied the effects triggered by molecular features that frequently find applications in plasmid design on dumbbell-driven gene expression: 1. A chimeric intron derived from the human β-globin gene–splicing is known to facilitate RNA processing, nuclear export, and subsequently gene expression (Luo and Reed, 1999); and 2. The Simian virus 40 (SV40) enhancer which can enhance the activity of the homologous SV40 promoter and which in addition was reported to function as an active DNA nuclear import sequence (Dean, 1997; Dean et al., 1999). The proposed mechanism behind this phenomenon is that the SV40 enhancer recruits transcription factors harboring protein nuclear import signals in the cytoplasm and that the vector DNA is piggyback translocated into the nucleus with support of the protein nuclear import machinery. We generated luciferase-expressing dumbbell vectors harboring either both, only one or none of these molecular features and monitored transgene expression in HEK293T and HepG2 cells. Our data demonstrate that introns and the SV40 enhancer can substantially improve dumbbell vector design (Jiang et al., 2016).
Materials and Reagents
Pipette tips (Corning® Isotip® filtered 0.2-10 μl) (Corning, catalog number: 4807 )
Pipette tips (Axygen® TF200RS 1-200 μl) (Corning, Axygen®, catalog number: TF-200-R-S )
Pipette tips (Axygen® TF1000RS 100-1,000 μl) (Corning, Axygen®, catalog number: TF-1000-R-S )
1.5 ml microcentrifuge tubes (RNase, DNase and Pyrogen-Free) (Corning, Axygen®, catalog number: MCT-150-C )
0.2 ml thin-walled PCR tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3412 )
FalconTM 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
T-75 flask (Corning, catalog number: 3290 )
24-well cell culture plate (Corning, catalog number: 3527 )
96-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 442404 )
HEK293T cells (ATCC, catalog number: CRL-3216 )
HepG2 cells (ATCC, catalog number: HB-8065 )
pGL3-control vector (Promega, catalog number: E1741 )
One Shot® TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404010 )
Chimeric human β-globin intron sequence (Gene synthesis, GeneArt, Applied Biosystems): 5’-CAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGACGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGG-3’
SV40 enhancer sequence: 5’-CGATGGAGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGC-3’
Oligonucleotide primers for chimeric intron amplification (Synthesized by Integrated DNA Technologies, 25 nmol scale, deprotected desalted):
intron-Fw: 5’-ATCTATCGGGATCCAAGCTTCAGGTAAGTATCAAGGTTACAAGACAGG-3’
intron-Rv: 5’-CATTATCTGGATCCCCATGGACCCTGTGGAGAGAAAGGCAA-3’
Loop sequences (Synthesized by Integrated DNA Technologies, 25 nmol scale, deprotected desalted):
Loop-1: 5’-pGATCTGACCAGTTTTCTGGTCA-3’
Loop-2: 5’-pTCGACAGGCTCTTTTGAGCCTG-3’
Oligonucleotide primers for poly(A) signal (Synthesized by Integrated DNA Technologies, 25 nmol scale, deprotected desalted):
polyA-Fw: 5’-TGTAATTCTAGAGTCGGGGCG-3’
polyA-Rv: 5’-ATCTATCGGGATCCTTACCACATTTGTAGAGGTT-3’
FastDigest HindIII (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0504 )
FastDigest NcoI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0573 )
FastDigest BamHI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0054 )
FastDigest BglII (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0083 )
FastDigest XhoI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0694 )
FastDigest SalI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0644 )
FastDigest AseI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0914 )
UltraPureTM DNase/RNase-free distilled water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
T7 DNA polymerase (10 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0081 )
Taq DNA polymerase, recombinant (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0402 )
10x FD buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B64 )
T4 DNA ligase (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EL0014 )
dNTP set 100 mM solutions (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0181 )
QIAquick PCR purification kit (QIAGEN, catalog number: 28106 )
Potassium acetate (Sigma-Aldrich, catalog number: P1190-100G )
Agarose, LE, analytical grade (Promega, catalog number: V3125 )
Ethanol, absolute (Fisher Scientific, catalog number: BP28184 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-500G )
Magnesium chloride hexachloride (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670-100G )
Tris-HCl (Roche Diagnostics, catalog number: 10812846001 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS-100G )
Adenosine 5’-triphosphate disodium salt hydrate (Sigma-Aldrich, catalog number: A2383-1G )
Ethidium bromide solution (Bio-Rad Laboratories, catalog number: 1610433 )
Phenol solution (Sigma-Aldrich, catalog number: P4557-100ML )
Chloroform (Sigma-Aldrich, catalog number: 288306-1L )
3-Methyl-1-butanol (Sigma-Aldrich, catalog number: 309435-100ML )
GeneRuler DNA Ladder Mix (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0331 )
HyClone Dulbecco’s modified Eagles medium/high glucose with L-glutamine, sodium pyruvate (GE Healthcare, HyCloneTM, catalog number: SH30243.01 )
HyClone Standard Fetal Bovine Serum (GE Healthcare, HyCloneTM, catalog number: SH30088.03 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
Opti-MEM® I Reduced Serum Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
Luciferase Assay System (Promega, catalog number: E1501 )
Liquid nitrogen
TE buffer (see Recipes)
Equipment
Pipettes (Gilson, PIPETMAN Classic®, P2 , P20N , P200N , and P1000N )
CO2 incubator (Thermo Electron)
Shaker (Heidolph Instruments, model: Unimax 2010 )
Glass beaker (Schott, Duran)
Standard thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: GeneAmp PCR System 9700 )
Note: This product has been discontinued.
Gel doc (Bio-Rad Laboratories, Gel Doc Imager)
Gel running apparatus (Amersham Biosciences)
Gel staining tray (GE Healthcare)
Benchtop centrifuge (Eppendorf, model: 5430 R )
Heat block (Thermomixer comfort) (Eppendorf)
Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
Microwave (Panasonic)
Class II Biological Safety Cabinet (Gelman)
Synergy H1 Multi-Mode Reader (BioTek Instruments, model: Synergy H1 )
Software
GraphPad Prism software 5.0
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Jiang, X. and Patzel, V. (2017). Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors. Bio-protocol 7(15): e2425. DOI: 10.21769/BioProtoc.2425.
Download Citation in RIS Format
Category
Microbiology > Microbial biochemistry > DNA
Molecular Biology > DNA > Gene expression
Molecular Biology > DNA > DNA cloning
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2,426 | https://bio-protocol.org/exchange/protocoldetail?id=2426&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Overrepresentation Analyses of Differentially Expressed Genes in the Smut Fungus Ustilago bromivora during Saprophytic and in planta Growth
AC Angelika Czedik-Eysenberg
FR Franziska Rabe
HE Heinz Ekker
CC Carmen Czepe
AD Armin Djamei
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2426 Views: 8304
Edited by: Arsalan Daudi
Reviewed by: Malou Fraiture
Original Research Article:
The authors used this protocol in Nov 2016
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Original research article
The authors used this protocol in:
Nov 2016
Abstract
We have established the Ustilago bromivora–Brachypodium spp. interaction as a new model pathosystem for biotrophic fungal plant infections of the head smut type (Rabe et al., 2016). In this protocol, the methodology used for comparing gene expression between saprophytic and in planta growth of the fungus is described. The experimental and analytical pipeline, how next generation RNA sequencing (Illumina RNA-Seq) analysis can be used to obtain lists of genes significantly up or down regulated in planta in comparison to axenic culture is given. Furthermore, different methods to identify functional categories that are over- or under-represented among specific classes of genes are presented.
Keywords: Plant infection Biotrophic plant pathogens Fungal pathogens Smuts Ustilago bromivora RNA-seq Differential expression over/under representation analysis
Background
RNA deep sequencing (RNA-Seq) is a powerful and versatile tool to gain insights into the responses of cells and organisms to environmental changes and their adaptations to new developmental stages. A striking change of life situation comes with the switch from yeast-like growth to filamentous, pathogenicity associated growth modes in non-obligate pathogens. We studied this switch in the biotrophic fungal plant pathogen Ustilago bromivora (Rabe et al., 2016). RNA-Seq from infected tissue is a special situation, since reads from both–the host and the pathogen–will be identified. Here necessary considerations are described. These include the sequencing depth required to sufficiently cover the pathogen in the host tissue, and the methods used to align and quantify the resulting mixed pool of reads. Over/underrepresentation analysis (ORA) is a method to link expression changes to potential biological responses by looking if certain classes of transcripts respond in a concerted way. Three methodologies are described that can be used to statistically test for over- or underrepresentation of classes of transcripts: The first method tests ORA individually for defined classes of interest, such as predicted secreted proteins, using Fisher exact test (example for R implementation given). The other two approaches are ‘explorative’ analyses that test over/underrepresentation across all functional classes defined in a given functional annotation framework (FunCat or Mapman annotation).
Materials and Reagents
Pipetman Diamond tips, D200 (Gilson, catalog number: F161931 )
Pipetman Diamond tips, D1000 (Gilson, catalog number: F161671 )
50 ml sterile disposable vial (SARSTEDT, catalog number: 62.547.254 )
1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
Micro-homogenizer (Carl Roth, catalog number: K994.1 )
Glycerol anhydrous (Applichem, catalog number: A1123,1000 )
Liquid nitrogen
Sodium hypochlorite ~10% (Honeywell International, catalog number: 71696-2.5L )
Hydrochloric acid (HCl) 37% (Applichem, catalog number: 131020.1211 )
TRIzol reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596026 )
TURBO DNA-free Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1907 )
Ribo-Zero rRNA Removal Kit (Plant) (Illumina, catalog number: MRZPL1224 )
NEB Next Ultra RNA Library Prep Kit (New England Biolabs, catalog number: E7530S )
Ampure XP beads (Beckman Coulter, catalog number: A63882 )
Agilent RNA 6000 Nano Kit for Bioanalyzer (Agilent Technologies, catalog number: 5067-1511 ) or Standard Sensitivity RNA Analysis Kit (15 nt) (Advanced Analytical Technologies, catalog number: DNF-471 )
Illumina PE Cluster Kit (Illumina, catalog number: FC-401-4003 )
Illumina 250 cycle SBS reagents (Illumina, catalog number: PE-401-4001 )
Potato dextrose broth (BD, catalog number: 254920 )
Standard potting soil (Topfsubstrat ED63, Einheitserde, catalog number: SP ED63 T, obtained from GBC-Gartenbauzentrum Schwechat, catalog number: 013224 )
Perlite (Perlite Premium 2-6 mm, Gramoflor, obtained from GBC-Gartenbauzentrum Schwechat, catalog number: 079568 )
Silica sand (Quarzsand 0.5-2 mm, min2C, obtained from GBC-Gartenbauzentrum Schwechat, catalog number: 005989 )
Germination soil (Aussaaterde, Huminsubstrat N3, Neuhaus, Klasmann-Deilmann, obtained from GBC-Gartenbauzentrum Schwechat, catalog number: 001318 )
Potato dextrose liquid medium (PD medium) (see Recipes)
Soil mix (see Recipes)
Equipment
Pipetman P1000 (Gilson, catalog number: F123602 )
Pipetman P200 (Gilson, catalog number: F123601 )
Centrifuge for 50 ml disposable vials (e.g., Eppendorf, model: 5810 R )
Spectral photometer capable of measuring OD at 600 nm
ND-1000 NanoDrop Spectrophotometer (Thermo Fisher Scientific, model: NanoDrop 1000 )
2100 Bioanalyzer (Agilent Technologies, model: 2100 Bioanalyzer ) or Fragment Analyzer 12 (Advanced Analytical Technologies, model: Fragment Analyzer 12)
Illumina HiSeq2500 instrument (Illumina, model: HiSeq® 2500 )
Fume hood
Software
FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) (freely available)
Cutadapt (Martin, 2011) (freely available)
Kallisto (Bray et al., 2016) (freely available)
R statistical environment (R Development Core Team, 2012) (freely available)
For those not familiar with R, the following resources may be helpful:
https://cran.r-project.org/doc/manuals/r-release/R-intro.pdf
https://onlinecourses.science.psu.edu/statprogram/r
https://www.youtube.com/watch?v=7cGwYMhPDUY
DESeq2 R package (Love et al., 2014) (freely available)
FunCat workflow (Ruepp et al., 2004) implemented on the Pedant home page (http://mips.helmholtz-muenchen.de/funcatDB/index_update.html) (freely available for publicly released species)
Mercator online tool (http://www.plabipd.de/portal/web/guest/mercator-sequence-annotation) (freely available)
Pageman tool (Usadel et al., 2006), part of the Mapman software suit: http://mapman.gabipd.org/web/guest/mapman (freely available)
Procedure
Obtain samples of saprophytically grown U. bromivora
To obtain samples of saprophytically grown U. bromivora, PD medium (see Recipes) was inoculated from a mating type 1 strain (UB1) glycerol stock kept at -80 °C. The glycerol stock was produced by mixing 1 ml UB1 overnight culture with 1 ml 50% glycerol. For the original isolation of U. bromivora spore lines please refer to (Bosch and Djamei, 2017).
Cell are grown at 21 °C and 200 rpm to an OD600 nm = ~0.8 in three independent biological replicates. Fungal material is sampled by centrifugation in 50 ml vials at 1,200 x g for 5 min. The supernatant is carefully poured off and the pellet is shock frozen in liquid nitrogen. If other species are grown, it can be the case that the pellet is less stable and the supernatant should be pipetted off.
Obtain samples of the fungus growing in planta
To obtain samples of the fungus growing in planta, caryopses of Brachypodium hybridum Bd28 are gas sterilized by putting them into a closed exicator together with two 50 ml tubes each with 25 ml 5% sodium hypochlorite solution + 0.75 ml 37% HCl for 2 h.
After gas sterilization, seeds are left in a 50 ml tube in a fume hood without cover for 1 h, so that any residual chlorine gas can evaporate.
The seeds are then soaked in sterile filtered tap water for 1-2 h, and germinated for 2-3 weeks at 4 °C in the dark.
The seedlings are subsequently infected with U. bromivora spore solution. The spore solution is obtained by grinding spore sori filled spikelets of infected plants in a small amount (e.g., 1 ml) of filtered tap water with a micro homogenizer in a disposable vial with a V-shaped bottom (e.g., 1.5 ml micro centrifuge tube). The seedlings are kept moistened (but not submerged) with spore solution at 4 °C for one week and are then planted in soil (see soil mix in the recipes section).
12 days after planting, plant stems are harvested and shock frozen in liquid nitrogen.
RNA extraction
a.RNA is extracted using TRIzol (Chomczynski and Sacchi, 2006) according to the manufacturer’s protocol.
b.Residual DNA is removed with the TURBO DNA-free Kit according to the manufacturer’s instructions.
Determination of RNA quality and quantity
RNA quality should be verified: To determine RNA concentration and purity, the RNA should be measured with a NanoDrop spectrophotometer. A 260:280 ratio close to 2 and a 260:230 ratio of approximately 2-2.2 is desirable. A minimal amount of 100-200 ng RNA is required for subsequent library preparation. To determine that the isolated RNA is not significantly degraded, a Bioanalyzer or Fragment Analyzer with appropriate kits can be used (see Equipment). Measurements are conducted according to the manufacturer’s instructions. The RNA integrity number (RIN) provided by the Bioanalyzer software is not applicable to the in planta samples, since they contain plant and fungal rRNAs (example: see Figure 1). However, the Bioanalyzer or Fragment Analyzer plots should be manually evaluated for the following criteria: All rRNA peaks should be narrow and near symmetrically shaped and the baseline between peaks should be flat and close to 0.
Alternatively, an RNA gel (1% agarose gel in TBE) can be used to assess RNA integrity, even though this method is less precise than using the Bioanalyzer/Fragment Analyzer. For high quality RNA samples from saprophytic growth two clear bands (rRNAs) and for in planta samples four bands should be seen, with almost no additional smear.
Figure 1. Bioanalyzer plot for a high quality RNA sample extracted from U. bromivora infected Brachypodium tissue
Library construction
Before library preparation ribosomal RNA is removed from the samples using Ribo-Zero rRNA Removal Kit following manufacturer instructions.
The libraries are prepared using the NEB Next Ultra RNA Library Prep Kit. Size selection is performed using Ampure XP beads. The Bioanalyzer/Fragment Analyzer is used to test the size distribution of the libraries, followed by qPCR to determine the correct concentration needed for cluster generation.
The libraries are sequenced in paired end mode (PE125), using an Illumina HiSeq2500 instrument.
Notes:
An important consideration, when deciding on the sequencing depth, is sufficient coverage of pathogen transcripts in libraries derived from infected host tissue (see Notes).
We sequenced the in planta samples to an average depth of 130,000,000 raw reads and the axenic culture samples to an average depth of 23,000,000 raw reads.
The quality of the resulting reads is assessed using FastQC: Properties examined are the per-base quality to exclude sequencing problems like quality drop-off towards the end or failed cycles, kmer-distribution and overrepresented sequences to identify adapter dimers and short inserts and duplication rate to exclude overamplification or other library problems. Except for low amounts of reads showing Illumina adapter sequences on the 3’ end we did not experience any obvious problems. The adapter sequences are removed from the reads using cutadapt v1.4.2.
Data analysis
Figure 2 shows a flow diagram of the different steps involved in RNA-Seq data analysis, indicating the software tools used.
Figure 2. Workflow of RNA-Seq data analysis. Dark blue background: Input from lllumina RNA sequencing; Light blue: Sequencing quality control and read quantification; Green: Identification of differentially expressed genes; Orange: Three methods for over/under-representation analysis.
Identification of differentially regulated genes
The trimmed RNA-Seq reads are then quantified against the combined transcriptomes extracted from Brachypodium distachyon Bd21 (Bdistachyon_283_v2.1) and Ustilago bromivora UB1 annotations, with the Kallisto software, using default parameters. This constitutes a pseudo-alignment against the transcriptome. In cases where more than one splicing variant encoded by the same gene is identified (in our dataset 20 cases), the splicing variants can be treated and counted as individual genes in all subsequent analyses.
Further analysis of the dataset can be conducted with the statistical environment R. For comparison of U. bromivora gene expression between the saprophytic and in planta growth conditions, only U. bromivora transcripts (i.e., those with a gene identifier starting with ‘UBRO’) are retained. Differential expression statistics are computed using the DESeq2 R package. To estimate the size factors between samples, the default assumption that the overall fungal expression levels are similar between all samples is used (estimateSizeFactors:type = ‘ratio’).
Transcripts are considered significantly up- or downregulated in planta, if the log2-fold-change compared to axenic culture is > 2/< -2 and the Benjamini-Hochberg (Hochberg and Benjamini, 1990) corrected P-value is < 0.1.
Additionally, to reliably assess downregulation in planta despite the lower coverage of fungal reads in the in planta samples, we required an average of at least 150 reads for a transcript in the axenic samples to consider it in planta downregulated. (See more details in the Notes section).
Over/Underrepresentation analysis
To test over-/underrepresentation (ORA) of transcript classes of interest among the in planta up- and downregulated transcripts, one of the following strategies can be used depending on the situation:
For user-defined lists of transcripts (e.g., in our case the list of transcripts encoding for predicted secreted proteins), over/underrepresentation of the given class among the lists of significantly responding transcripts compared to the representation of the class among all predicted or expressed transcripts can be calculated using Fisher exact test in the R statistical environment. The following code can be used:
input <- cbind(total=c(Nrtotal_secreted, Nrtotal non-secreted), in_planta_up=c(Nrin-planta up secreted, Nrin-planta up non-secreted))
fisher.test(input)
In the fisher.test() function the parameter alternative=”greater” or alternative=”less” can be added to test for only overrepresentation or only underrepresentation in cases where one of the two options can be precluded.
Nrtotal_secreted .. the number of transcripts encoding for predicted secreted proteins among all predicted or expressed transcripts
Nrtotal_non-secreted .. the number of transcripts encoding for predicted non-secreted proteins among all predicted or expressed transcripts
Nrin-planta up secreted .. the number of transcripts encoding for predicted secreted proteins among those transcripts identified to significantly upregulated in-planta compared to saprophytic growth
Nrin-planta up non-secreted .. the number of transcripts encoding for predicted non-secreted proteins among those transcripts identified to significantly upregulated in-planta compared to saprophytic growth.
To systemically test for overrepresentation of functional classes among the transcripts significantly up or down regulated in planta, the available FunCat classification and ORA workflow for the predicted U. bromivora transcripts http://mips.helmholtz-muenchen.de/funcatDB/index_update.html can be used.
Alternatively, the following workflow can be used for all species, also when FunCat annotation is not available:
Use of the Mercator online tool to generate a functional annotation in the Mapman format–predicted transcripts have to be submitted in FASTA format.
Use of the Pageman tool (integrated in the Mapman software suit http://mapman.gabipd.org/web/guest/mapman) to conduct ORA. We used Fisher test statistics and the p-values were corrected for multiple testing by the Benjamini-Hochberg algorithm.
Notes
All sequencing raw data (.bam files) as well as counts of each U. bromivora gene in each sample from our experiment published in Rabe et al. (2016) can be downloaded from GeneExpressionOmnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE87751.
Considerations concerning sequencing depth of samples from pathogen infected host tissue: The required sequencing depth will depend on both, the proportion of pathogen RNA compared to host RNA and the expected expression level of genes of interest. These properties may vary, depending on the time point during the infection cycle and the tissue studied. A quantitative real time (qRT)-PCR pre-assay to compare host against pathogen genomic marker can help to evaluate the ratio between host versus pathogen cells.
Below we give some examples from our dataset that may help to estimate relevant properties and asses the reliability of expression differences observed for individual genes:
Despite obtaining on average in total approximately 103,500,000 map-able reads in the in planta samples and only 13,800,000 in the axenic culture samples, i.e., ~7.5x more total reads, the in planta samples contain only an average of ~175,000 U. bromivora reads, compared to ~14,000,000 fungal reads in the samples of fungus grown in axenic culture. We thus obtained an approximately 80x better coverage of the axenic stage. This corresponds to having ~600x more plant reads compared to fungal reads in the mixed samples, a similar ratio to what we get when directly comparing the plant and fungal reads in the mixed samples. Having 80x lower coverage means that a transcript expressed equally in axenic culture and in planta that obtains 80 reads in axenic culture would typically obtain only one read in planta. Therefore, to reliably assess whether a transcript for which no reads are found in planta is downregulated compared to axenic culture, sufficient reads are required in the axenic culture sample. We defined a threshold of at least 150 reads (roughly 2x the difference in coverage) in axenic culture, additional to a significant P-value for the DESeq2 analysis, to consider a transcript for which no reads were obtained in planta as significantly downregulated in planta. Applying these filters, we identified 888 U. bromivora transcripts for which downregulation in planta could not be reliably assessed due to insufficient coverage.
Suppliers and order numbers given for chemicals, consumables and instruments refer to the equipment used by us. In general, it can be exchanged for equivalent equipment from other suppliers.
Recipes
Potato dextrose liquid medium (PD medium)
24 g/L potato dextrose broth in deionized water
Autoclaved
Soil mix
3:1:1:1 standard potting soil:perlite:silica sand:germination soil
Acknowledgments
This protocol is adapted from Rabe et al. (2016). We would like to thank all the people involved in the works for this protocol as well as the original publication that it is based upon. The research leading to these results received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. [EUP0012 ‘Effectomics’], the Austrian Science Fund (FWF): [P27429-B22, P27818-B22, I3033-B22], and the Austrian Academy of Science (OEAW).
References
Bosch, J. and Djamei, A. (2017). Isolation of Ustilago bromivora strains from infected spikelets through spore recovery and germination. Bio Protoc 7(14): e2392.
Bray, N. L., Pimentel, H., Melsted, P. and Pachter, L. (2016). Near-optimal probabilistic RNA-seq quantification.v Nat Biotechnol 34: 525-527.
Chomczynski, P and Sacchi, N. (2006). The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc 1(2): 581-585.
Hochberg, Y and Benjamini, Y. (1990). More powerful procedures for multiple significance testing. Stat Med 9(7): 811-818.
Lohse, M., Nagel, A., Herter, T., May, P., Schroda, M., Zrenner, R., Tohge, T., Fernie, A. R., Stitt, M. and Usadel, B. (2014). Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ 37(5): 1250-1258.
Love, M. I., Huber, W. and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12): 550.
Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet 17: 10-12.
Rabe, F., Bosch, J., Stirnberg, A., Guse, T., Bauer, L., Seitner, D., Rabanal, F. A., Czedik-Eysenberg, A., Uhse, S., Bindics, J., Genenncher, B., Navarrete, F., Kellner, R., Ekker, H., Kumlehn, J., Vogel, J. P., Gordon, S. P., Marcel, T. C., Münsterkötter, M., Walter, M. C., Sieber, C. M., Mannhaupt, G., Güldener, U., Kahmann, R. and Djamei, A. (2016). A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem. eLife 5: 179-188.
R Development Core Team. (2012). R: A language and environment for statistical computing. R Foundation for Statistical Computing.
Ruepp, A., Zollner, A., Maier, D., Albermann, K., Hani, J., Mokrejs, M., Tetko, I., Güldener, U., Mannhaupt, G., Münsterkötter, M. and Mewes, H. W. (2004). The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes. Nucleic Acids Res 32(18): 5539-5545.
Usadel, B., Nagel, A., Steinhauser, D., Gibon, Y., Bläsing, O. E., Redestig, H., Sreenivasulu, N., Krall, L., Hannah, M. A., Poree, F., Fernie, A. R and Stitt, M. (2006). PageMan: an interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments. BMC Bioinformatics 7: 535.
Copyright: Czedik-Eysenberg et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Czedik-Eysenberg, A. B., Rabe, F., Ekker, H., Czepe, C. and Djamei, A. (2017). Overrepresentation Analyses of Differentially Expressed Genes in the Smut Fungus Ustilago bromivora during Saprophytic and in planta Growth. Bio-protocol 7(15): e2426. DOI: 10.21769/BioProtoc.2426.
Rabe, F., Bosch, J., Stirnberg, A., Guse, T., Bauer, L., Seitner, D., Rabanal, F. A., Czedik-Eysenberg, A., Uhse, S., Bindics, J., Genenncher, B., Navarrete, F., Kellner, R., Ekker, H., Kumlehn, J., Vogel, J. P., Gordon, S. P., Marcel, T. C., Münsterkötter, M., Walter, M. C., Sieber, C. M., Mannhaupt, G., Güldener, U., Kahmann, R. and Djamei, A. (2016). A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem. eLife 5: 179-188.
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Category
Plant Science > Plant immunity > Host-microbe interactions
Plant Science > Plant immunity > Perception and signaling
Molecular Biology > DNA > Gene expression
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2,427 | https://bio-protocol.org/exchange/protocoldetail?id=2427&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Membrane Lipid Screen to Identify Molecular Targets of Biomolecules
JJ John R. Jimah
PS Paul H. Schlesinger
NT Niraj H. Tolia
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2427 Views: 8464
Original Research Article:
The authors used this protocol in Dec 2016
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Original research article
The authors used this protocol in:
Dec 2016
Abstract
Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al., 2016). This protocol has a companion protocol: ‘Liposome disruption assay to examine lytic properties of biomolecules’ which can be applied to examine the ability of the biomolecule to disrupt membranes composed of the lipid target identified by following this protocol (Jimah et al., 2017).
Keywords: Membrane Liposome Lipid Disruption Lysis Carboxyfluorescein Leakage Specificity
Background
Proteins and biomolecules with membrane disruption activities, such as pore formation or membrane fusion, may target specific lipids within membranes. Examples of lipid-specific pore-formation include Plasmodium CelTOS that depends on phosphatidic acid for pore formation, and the cholesterol dependent cytolysins (Jimah et al., 2016; Lukoyanova et al., 2016). CelTOS (cell traversal protein for ookinetes and sporozoites) is a malaria parasite protein that disrupts host cell membranes by pore formation to enable the exit of parasites from invaded host cells during cell traversal (Kariu et al., 2006; Jimah et al., 2016). Cholesterol dependent cytolysins are a large class of pore-forming proteins, including virulence factors of gram positive bacteria such as pneumolysin and listeriolysin (Lukoyanova et al., 2016). Identifying the specific lipids targeted informs the mechanism of membrane disruption that underlies biological function and role of proteins and biomolecules.
Materials and Reagents
Small gel incubation tray (Santa Cruz Biotechnology, catalog number: sc-358889 )
Serological pipets, 10 ml (Genesee Scientific, catalog number: 12-104 )
Pipette tips
10 µl tips (VWR, catalog number: 46620-318 )
200 µl tips (VWR, catalog number: 53509-009 )
1,000 µl tips (VWR, catalog number: 83007-384 )
Centrifuge tubes, 50 ml (Genesee Scientific, catalog number: 21-106 )
Microcentrifuge tubes, 1.7 ml (MIDSCI, catalog number: AVSS1700RA )
Membrane lipid strips with dimensions 2 x 3 cm (Echelon Biosciences, catalog number: P-6002 )
Protein or biomolecule of interest
Tris (Gold Bio, catalog number: T-400-5 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-25KG )
Tween 20% (Sigma-Aldrich, catalog number: P1379 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906-500G )
Primary antibody against the tagged protein or biomolecule of interest
Peroxidase conjugated secondary antibody
ECL Prime Western Blotting Detection Kit (GE Healthcare, catalog number: RPN2232 )
Phospholipids (dissolved in chloroform, commonly used phospholipids are):
DOPC (Avanti Polar Lipids, catalog number: 850375C )
POPC (Avanti Polar Lipids, catalog number: 850457C )
POPA (Avanti Polar Lipids, catalog number: 840857C )
POPS (Avanti Polar Lipids, catalog number: 840034C )
Blocking buffer (see Recipes)
Wash buffer (see Recipes)
Note: See the ‘Notes’ section for a list of materials and reagents used in the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ that is recommended for follow up experiments (Jimah et al., 2017).
Equipment
Pipetman Classic pipets
P10 (Gilson, catalog number: F144802 )
P20 (Gilson, catalog number: F123600 )
P200 (Gilson, catalog number: F123601 )
P1000 (Gilson, catalog number: F123602 )
Pipet-Aid XP Pipette controller (Drummond Scientific, catalog number: 4-000-101 )
Incubator
BenchRockerTM variable 2D rocker (Benchmark Scientific, catalog number: BR2000 )
pH meter (Fisher Scientific, model: AccumetTM AE150, catalog number: 13-636-AE153 )
Fluorescent image analyzer (Fujifilm, model: FLA-5000 )
Note: See the ‘Notes’ section for a list of equipment used in the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ that is recommended for follow up experiments (Jimah et al., 2017).
Procedure
Screen to identify specific membrane lipid binding by biomolecules of interest
Purify or obtain the biomolecule of interest.
Note: Ensure the protein is tagged, for example with a 6-His tag.
Place lipid strips in a small gel incubation tray, and block lipid strips with 10 ml blocking buffer (see Recipes) at room temperature for one hour.
Notes:
See the ‘Notes’ section for a description of the lipid component of the lipid strips.
The buffer and pH used depend on the biomolecule tested. For example, acidic conditions could be used if the biomolecule functions in an acidic environment. Also, use enough buffer to cover the lipid strip.
Wash three times, for five minutes each, with 10 ml wash buffer (see Recipes).
Note: Suggestions on the duration of washing, blocking and incubation times is described in the Notes section.
Incubate the membrane lipid strips with the protein or biomolecule in 10 ml blocking buffer for one hour at room temperature.
Note: The concentration of protein used is determined empirically, and is usually at the low micromolar to nanomolar concentrations.
Wash three times, for five minutes each, with 10 ml wash buffer.
Incubate the membrane lipid strips with a primary antibody against the tagged protein or biomolecule in 10 ml blocking buffer for one hour at room temperature.
Notes:
The primary antibody could be against the protein tag or specific to the protein or biomolecule.
The concentration of primary antibody used is specified by the manufacturer.
Wash three times, for five minutes each, with 10 ml wash buffer.
Incubate the membrane lipid strips with a peroxidase conjugated secondary antibody, against the primary antibody, in 10 ml blocking buffer for one hour at room temperature.
Note: The concentration of secondary antibody used is specified by the manufacturer.
Use the ECL Prime Western Blotting Detection Kit to detect the peroxidase conjugated secondary antibody.
Image the chemiluminescence using a fluorescent image analyser (Figure 1).
Figure 1. Lipid strips probed with PfCelTOS or controls. A. Lipid strip probed with PfCelTOS, followed by primary antibody to CelTOS and a secondary antibody conjugated with peroxidase for visualization. PfCelTOS specifically bound to phosphatidic acid (Jimah et al., 2016). B. Lipid strip probed with primary antibody against His-tag and a secondary antibody conjugated with peroxidase for visualization. Neither the primary or secondary antibody bound to the lipid strip.
Data analysis
Screen to identify specific membrane lipid binding by biomolecules of interest:
Spot intensities in each strip are normalized to the background for that strip and to the intensities in the negative control(s).
Note: The negative control may be a no protein control, or a protein that does not bind lipids.
The normalized spot intensities, from experimental replicates, are compared to determine if there is a statistical preference for specific lipids by one-way ANOVA.
Note: Perform appropriate number of replicates for statistical analysis. An example of representative data and analysis is reported in Jimah et al., 2016.
Conclusion
The protocol described here enables the identification of specific lipids that are targeted by proteins of biomolecules for binding. In addition to binding lipid molecules, some proteins or biomolecules may disrupt membranes by targeting a specific lipid component of a membrane. A companion protocol: ‘Liposome disruption assay to test lytic properties of biomolecules’ describes how to determine and quantify the disruption of membranes by proteins or biomolecules (Jimah et al., 2017). Below is a summary of ‘Screen to identify membrane lipids targeted for membrane disruption by biomolecules’.
Procedure: Screen to identify membrane lipids targeted for membrane disruption by biomolecules
Notes:
The identification of specific lipids targeted for binding by biomolecules, described above, will inform the lipids targeted for membrane disruption since binding precedes membrane disruption.
The protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ will be applied to determine if the presence of a particular lipid within membranes is necessary for membrane disruption.
Make liposomes composed of lipids that have been identified to bind the biomolecule of interest.
Note: For example, if the biomolecule binds phosphatidic acid, liposomes composed of phosphatidylcholine and phosphatidic acid at 8:2 molar ratio may be made. The addition of phosphatidylcholine is because liposomes composed of phosphatidic acid alone are unstable. In the case of this example, it is also useful to make liposomes composed only of phosphatidylcholine to serve as a negative control.
Investigate the membrane disruption activity of the biomolecule on the liposomes following the protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’.
Test a range of concentrations of the biomolecule for the ability to disrupt liposomes.
Note: Recommended concentrations are within the nanomolar and micromolar range. The biomolecule targets specific lipids if low nanomolar concentrations of the biomolecule disrupt membranes containing particular lipids. It may be possible that at high concentrations, the biomolecule nonspecifically disrupts membranes composed of other lipids.
Data analysis: Screen to identify membrane lipids targeted for membrane disruption by biomolecules
Note: Please refer to the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ for a detailed description of data analysis. (Jimah et al., 2017).
Notes
Membrane lipid strips (Echelon Biosciences) contain the common membrane lipids: triglyceride, phosphatidylinositol, phosphatidylinositol (4)-phosphate, phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, diacylglycerol, cholesterol, phosphatidylcholine, sphingomyelin, phosphatidylglycerol, 3-sulfogalactosylceramide and cardiolipin.
Another membrane lipid strip contains additional lipids that may be of interest (Echelon Biosciences, catalog number: P-6001) contains: Lysophosphatidic acid (LPA), Lysophosphocholine (LPC), Phosphatidylinositol (PtdIns), Phosphatidylinositol (3) phosphate (PtdIns(3)P), Phosphatidylinositol (4) phosphate (PtdIns(4)P), Phosphatidylinositol (5) phosphate (PtdIns(5)P), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingosine 1-Phosphate (S1P), Phosphatidylinositol (3,4) bisphosphate (PtdIns(3,4)P2), Phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), Phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2), Phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3), Phosphatidic acid (PA), Phosphatidylserine (PS).
The blocking, incubation, and washing times stated here are recommendations, and may be modified based on empirical observations.
Please refer to the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ for a detailed description of the materials, reagents, equipment and protocol necessary for the identification of membrane lipids targeted for membrane disruption by biomolecules (Jimah et al., 2017).
Recipes
Blocking buffer
10 mM Tris pH 8.0
150 mM NaCl
0.1% Tween 20%
3% BSA
Wash buffer
10 mM Tris pH 8.0
150 mM NaCl
0.1% Tween 20%
Acknowledgments
This work was supported by the Burroughs Wellcome Fund (to NHT) and National Institutes of Health (R56 AI080792 to NHT). This protocol was adapted from Jimah et al., 2016.
References
Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
Jimah, R. J., Schlesinger, H. P. and Tolia, H. N. (2017). Liposome disruption assay to examine lytic properties of biomolecules. Bio Protoc 7(15): e2433.
Kariu, T., Ishino, T., Yano, K., Chinzei, Y. and Yuda, M. (2006). CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts. Mol Microbiol 59(5): 1369-1379.
Lukoyanova, N., Hoogenboom, B. W. and Saibil, H. R. (2016). The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins. J Cell Sci 129(11): 2125-2133.
Copyright: Jimah et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Jimah, J. R., Schlesinger, P. H. and Tolia, N. H. (2017). Membrane Lipid Screen to Identify Molecular Targets of Biomolecules. Bio-protocol 7(15): e2427. DOI: 10.21769/BioProtoc.2427.
Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
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Category
Biochemistry > Protein > Activity
Microbiology > Microbe-host interactions > In vitro model
Biochemistry > Lipid > Lipid-protein interaction
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2,428 | https://bio-protocol.org/exchange/protocoldetail?id=2428&type=0 | # Bio-Protocol Content
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Peer-reviewed
Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species
YG Yvetta Gbelska
NH Nora Toth Hervay
VD Vladimira Dzugasova
AK Alexandra Konecna
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2428 Views: 7950
Edited by: Yanjie Li
Reviewed by: Lip Nam LOHSadri Znaidi
Original Research Article:
The authors used this protocol in Dec 2016
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Dec 2016
Abstract
Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an energy-dependent manner. Incubation of yeast cells in the presence of 2-deoxy-D-glucose (inhibitor of glycolysis) and R6G (mitochondrial ATPase inhibitor) leads to marked depletion of intracellular ATP pool (Kolaczkowski et al., 1996). An active KlPdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer.
Keywords: Rhodamine 6G Fluorescence Kluyveromyces lactis ABC transporter Transport activity assay
Background
Multidrug efflux pumps are widely distributed and can be found in all living species. They represent an important mechanism of antimicrobial resistance. The ability to quantify the activity of efflux pumps is necessary for understanding of their contribution to physiological processes and assessment of the validity of potential therapeutics (e.g., efflux inhibitors) (Blair and Piddock, 2016). Methods for efflux activity measurements largely rely on two different mechanisms. Some methods directly measure the substrate efflux, i.e., how much of the substrate is pumped out, and others measure substrate molecule accumulation inside the cell, the levels of which is then used to infer efflux indirectly. However, the latter is less sensitive due to variable membrane permeability that alters dye influx rates (Blair and Piddock, 2016). Accumulation of R6G in growing C. albicans cells inversely correlates with the level of the ABC transporter Candida drug resistance 1 (CDR1) mRNA expression, establishing levels of intracellular R6G accumulation can be therefore used for identification of azole-resistant strains (Maesaki et al., 1999). Historically, this was carried out by measurements of accumulated radiolabelled-substrates. More recently, fluorescence-based methods are being used. Accumulation of fluorescent dye in a single cell can also be measured by flow cytometry. The benefit of this approach lies in the ability to measure variation in efflux activity among individual cells.
The protocol of the above described method involves preloading the cell population with a fluorescent substrate prior to the efflux assay. After the loading step, substrate accumulates within the cells at maximum concentration. Cells are then washed to remove the substrate. Subsequently, glucose is supplemented to the culture as a source of energy, and the fluorescence signal of substrate is monitored. The method is suitable for use with any yeast species (Borecka-Melkusova et al., 2008).
Materials and Reagents
Pipettes tips
Sterile inoculation loop
50-ml polypropylene centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050 )
1.5-ml microcentrifuge tubes
96-well flat bottom with lid MicroWell plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167008 )
Yeast strain to be analysed
Yeast extract (Biolife Italiana, catalog number: 4122202 )
Bacto peptone (Biolife Italiana, catalog number: 4122592 )
HEPES, free acid (AMRESCO, catalog number: 0511 )
Sodium hydroxide (NaOH), 1 mol/L (Merck, catalog number: 109137 )
2-deoxy-D-glucose (Sigma-Aldrich, catalog number: D6134 )
Rhodamine 6G (Sigma-Aldrich, catalog number: R4127 )
D-glucose (Biolife Italiana, catalog number: 4125012 )
YEPD rich growth medium (see Recipes)
20 mM glucose (see Recipes)
50 mM HEPES/NaOH assay buffer (see Recipes)
2-deoxy-D-glucose in HEPES/NaOH buffer (see Recipes)
10 mM rhodamine 6G (see Recipes)
Equipment
Pipettes
Xplorer® 15-300 µl (Eppendorf, catalog number: 4861000031 )
Research® plus 2-0 µl (Eppendorf, catalog number: 3120000038 )
pH meter (Xylem, WTW, model: inoLab® pH 7110 )
Incubation shaker Unitron (Infors, model: Plus AJ252 )
Haemocytometer
Centrifuge 5804R (Eppendorf, model: 5804 R )
Centrifuge Mikro 200R (Hettich Lab Technology, model: MIKRO 200 R )
Spectrofluorometer Varioscan Flash (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5250500 )
Autoclave
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Gbelska, Y., Toth Hervay, N., Dzugasova, V. and Konecna, A. (2017). Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species. Bio-protocol 7(15): e2428. DOI: 10.21769/BioProtoc.2428.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Fluorescence
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2,429 | https://bio-protocol.org/exchange/protocoldetail?id=2429&type=0 | # Bio-Protocol Content
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Mouse Müller Cell Isolation and Culture
XL Xiao Liu
LT Luosheng Tang
YL Yongqing Liu
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2429 Views: 11284
Reviewed by: Karthik Krishnamurthy
Original Research Article:
The authors used this protocol in Jun 2017
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Abstract
Müller cells are the major supportive and protective glial cells across the retina. Unlike in fish, they have lost the capacity to regenerate the retina in mammals. But, mammalian Müller cells still retain certain retinal stem cell properties with various degree of self-renewal and differentiation potentials, and thereby held a merit in cell-based therapies for treating retinal degeneration diseases. In our laboratory, we use an enzymatic procedure to isolate, purify, and culture mouse Müller cells.
Keywords: Mouse Müller glial cells Stem cells Retinal cell isolation Primary cell culture Worthington Papain Kit
Background
Müller glial cell is a major lineage in the retina that functions to maintain retinal homeostasis through synthesis of neurotrophic factors, uptake and recycle of neurotransmitters, spatial buffering of ions, and maintenance of the blood-retinal barrier (Bringmann et al., 2006; De Melo Reis et al., 2008). Müller glia serve as retinal progenitor/stem cells in fish and, to a limited extent, in birds (Vihtelic and Hyde, 2000; Fischer and Reh, 2001). But, mammalian Müller cells have lost such a capacity to regenerate the retina, though still retain certain properties of adult stem cells such as proliferation upon a retinal damage. Researches in restoring of the lost capacity of mammalian Müller cells to repair retinal damage and understanding of the underlying mechanism are undertaken in laboratories with primary cells isolated from model animal retinas. Proteolytic enzymes are widely used in Müller cell dissociation and papain is less damaging and more effective than other proteases. Sarthy and Lam developed a method for dissociation and separation of glial cells with papain digestion followed by gentle mechanical dissociation, they found that among the enzymes used for dissociating turtle retina, papain produced the least trauma (Sarthy and Lam, 1978).
Materials and Reagents
Cell culture dishes: 35 x 10 mm (Corning, catalog number: 430166 )
15 ml centrifuge tubes (Corning, catalog number: 430790 )
Tipone® pipette tips (USA Scientific, catalog number: 1126-7810 )
5 ml pipets (Corning, Falcon®, catalog number: 357543 )
2 ml cryopreservation vials (Corning, catalog number: 430659 )
Alcohol Prep Pads (PDI, catalog number: B33905 )
Animals: 2- to 4-week-old mice
Worthington Papain Kit (papain dissociation system) (Worthington Biochemical, catalog number: LK003150 )
Note: The components of kit include vial 1 (Sterile Earle’s Balanced Salt Solution, EBSS), vial 2 (Papain containing L-cysteine and EDTA), vial 3 (Deoxyribonuclease I, DNase), and vial 4 (Ovomucoid protease inhibitor with bovine serum albumin).
70% ethanol
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-100TAB )
Penicillin-streptomycin (Pen/Strep) (10,000 μg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Cellgro®, catalog number: 10-013-CV )
Fetal bovine serum (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30071.03 )
Gelatin (Sigma-Aldrich, catalog number: G1890-100G )
0.25% trypsin ethylenediaminetetraacetic acid (EDTA) solution (Mediatech, Cellgro®, catalog number: 25-053-CI )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
Cell culture medium (see Recipes)
0.1% gelatin solution (see Recipes)
Cell freezing medium (see Recipes)
Equipment
Pipettes (Eppendorf)
Pipet-aid (Drummond)
Single Edge Blade (Sparco, catalog number: SPR11820 )
Dissection forceps and scissors
Iris Scissors Sharp Straight (Storz Ophthalmic Instruments, catalog number: E3344 )
Castroviejo Suturing Forceps 0.12 mm (Storz Ophthalmic Instruments, catalog number: E1796 )
37 °C, 5% CO2 cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: Model 3250 )
37 °C incubator (VWR, model: 1545 )
Allegra bench-top centrifuge (Beckman Coulter, model: Allegra® X-15R )
Lab quake rotisserie shaker (Barnstead Thermolyne LabQuake, model: 4152110 )
Inverted routine microscope (Nikon Instruments, model: Eclipse TS100 )
Stereo binocular microscope (Olympus, model: SZ40 )
Note: Mice are euthanized by carbon dioxide asphyxiation, a carbon dioxide source, regulated dispenser, and euthanasia chamber. All animal manipulations are conducted in accordance with the policies and guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) were approved by the University of Louisville, Louisville, Kentucky, USA.
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Liu, X., Tang, L. and Liu, Y. (2017). Mouse Müller Cell Isolation and Culture. Bio-protocol 7(15): e2429. DOI: 10.21769/BioProtoc.2429.
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Category
Stem Cell > Adult stem cell > Glial Stem Cell
Cell Biology > Cell isolation and culture > Cell differentiation
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243 | https://bio-protocol.org/exchange/protocoldetail?id=243&type=0 | # Bio-Protocol Content
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Stable Interference by shRNA with pSUPER.retro Vectors and Lipofectamine
SS Silvia Soddu
Published: Vol 2, Iss 15, Aug 5, 2012
DOI: 10.21769/BioProtoc.243 Views: 13584
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Abstract
RNA interference is a powerful genetic approach for efficiently silencing target genes. Expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes. This protocol describes a method to stably integrate shRNA constructs with pSUPER.retro vectors and Lipofectamine in RKO and H1299 cells. This method can be applied to cells from other lines with modification of drug selection and cell conditions. pSUPER.retro vectors can be also transfected into packaging cells by this same method to produce retroviral supernatants.
Keywords: Interference ShRNA Transfection
Materials and Reagents
Lipofectamine/plus transfection reagents (Life Technologies, Invitrogen™, catalog number: 11514-015 )
Dulbecco’s modification of eagles medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 21885025 )
Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25300-054 )
FBS (Life Technologies, Invitrogen™, catalog number: 10099-141 )
Puromycin (Sigma-Aldrich, catalog number: P8833 )
DPBS (Lonza, catalog number: BE17-512F )
pSUPER.retro.puro vector (OligoEngine, catalog number: VEC-PRT-0002 )
Equipment
Tissue culture hood
Tissue culture incubator
Culture dish (BD Biosciences, Falcon®: 353003 )
10 cm dish
Procedure
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Category
Molecular Biology > RNA > RNA interference
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2,430 | https://bio-protocol.org/exchange/protocoldetail?id=2430&type=0 | # Bio-Protocol Content
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Purification of FLAG-tagged Secreted Proteins from Mammalian Cells
EI Eisuke Itakura
CC Changchun Chen
MB Mario de Bono
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2430 Views: 10576
Edited by: Jyotiska Chaudhuri
Reviewed by: Liang Liu
Original Research Article:
The authors used this protocol in 31-Jan 2017
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Abstract
This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays.
Keywords: Secreted protein Purification Mammalian Ligand Receptor
Background
E. coli is one of the organisms of choice for production of recombinant proteins from both prokaryotes and eukaryotes. The major advantages of bacterial expression systems are high productivity and low cost. However, mature proteins are essential for functional analyses (e.g., ligand binding assays). Most secreted eukaryotic proteins undergo post-translational modification by covalent glycan linkage and formation of disulfide bonds in the endoplasmic reticulum. These covalent modifications are essential for the general stability and folding of the secreted mature proteins. Therefore, the best method for production of mammalian secreted proteins is the use of a mammalian host to ensure the production of recombinant proteins that have undergone proper post-translational modifications. In the following protocol, we have described the detailed procedure for purification of secreted proteins from mammalian cells. This protocol is for production of FLAG-tagged proteins, but should be applicable to that of other tagged (e.g., His-tag) proteins as well.
Materials and Reagents
Pipette tips (VWR)
12-well plate (Corning, Falcon®, catalog number: 353043 )
100 mm tissue culture dishes (Corning, Falcon®, catalog number: 353803 )
Amicon Ultra 15 filtration units 3K (EMD Millipore, catalog number: UFC900308 )
1.5 ml tubes (VWR, catalog number: 89000-028 )
50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
1 ml syringes (Terumo Medical, catalog number: SS-01T )
30 G needles (BD, catalog number: 305128 )
Flp-In T-rex 293 cell line (Thermo Fisher Scientific, InvitrogenTM, catalog number: R78007 ) (Optional)
pcDNA5 FRT TO (Thermo Fisher Scientific, InvitrogenTM, catalog number: V652020 ) (Optional)
Hygromycin B (NACALAI TESQUE, catalog number: 07296-11 ) (Optional)
Blasticidin (Wako Pure Chemical Industries, catalog number: 029-18701 ) (Optional)
Doxycycline (Takara Bio, Clontech, catalog number: 631311 ) (Optional)
Dulbecco’s modified eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
Fetal bovine serum (FBS) (Biosera, catalog number: FB-1345/500 )
Anti-Flag M2 agarose beads (Sigma-Aldrich, catalog number: A2220 )
Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
Potassium acetate (KAc) (Sigma-Aldrich, catalog number: 236497 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
3xFlag peptide (Sigma-Aldrich, catalog number: F4799 )
Phosphate buffered saline (PBS)
Growth medium (see Recipes)
Wash buffer (see Recipes)
Elution buffer (see Recipes)
Equipment
Pipettes (e.g., Gilson)
37 °C, 5% CO2 incubator (e.g., Panasonic Biomedical, catalog number: MCO-170AICUV-PE )
Tube rotator located in cold room (e.g., Fisher Scientific, catalog number: 88-861-049 )
Centrifuge (e.g., Eppendorf, model: 5804 )
Cell culture microscope (e.g., Carl Zeiss, Vienna, Austria)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Itakura, E., Chen, C. and de Bono, M. (2017). Purification of FLAG-tagged Secreted Proteins from Mammalian Cells. Bio-protocol 7(15): e2430. DOI: 10.21769/BioProtoc.2430.
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Category
Biochemistry > Protein > Isolation and purification
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2,431 | https://bio-protocol.org/exchange/protocoldetail?id=2431&type=0 | # Bio-Protocol Content
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Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System
MM Miriam Manor
Udi Qimron
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2431 Views: 10564
Edited by: Modesto Redrejo-Rodriguez
Original Research Article:
The authors used this protocol in 0 2014
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Abstract
We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages. This protocol allows seamless deletion of desired genes in a T7 phage, and can be expanded to other phages and other types of genetic manipulations as well.
Keywords: Bacteriophage Escherichia coli Homologous recombination Positive selection
Background
Bacteriophages (phages) are the most prevalent and widely distributed biological entity in the biosphere, highlighting their ecological importance (Suttle, 2007). Many studies also propose using phages for medical purposes (Weber-Dabrowska et al., 2001; Merril et al., 2003; Harper and Enright, 2011; Edgar et al., 2012; Bikard et al., 2014; Citorik et al., 2014; Yosef et al., 2014 and 2015). Unfortunately, only a few published methods detail genetic engineering of phage genomes (Selick et al., 1988; Marinelli et al., 2008; Pires et al., 2016) and in addition, some of these methods are tedious, and some cannot achieve desired results such as seamless deletions. A simple and efficient technique for seamless genetic engineering of phages is thus desired. In this protocol, we present a technique described in 2014 (Kiro et al., 2014) for deleting genes of the E. coli phage, T7. We first designed genetic constructs that facilitate desired homologous recombination events. We then used the CRISPR-Cas type I-E system to select desired engineered phages. Several other studies have also reported the use of CRISPR-Cas systems to engineer phages and the reader is referred to them for selecting the most appropriate and fitting protocols for the specific requirements (Martel and Moineau, 2014; Box et al., 2015; Lemay et al., 2017).
Materials and Reagents
Materials
1.7 ml microfuge tube (Corning, Axygen®, catalog number: MCT-175-C )
15 ml tube (Corning, catalog number: 430052 )
PCR tubes (Corning, Axygen®, catalog number: PCR-0208-C )
Pipette tips
Bacterial strains
Electro-competent BL21-AI (Invitrogen, Genotype: F− ompT hsdSB(rB−, mB−) gal dcm araB::T7RNAP-tetA, tetr)
Electro-competent NEB5α (New England Biolabs)
Phage
WT T7 phage (laboratory collection. Available at ATCC, catalog number: BAA-1025-B2 )
Plasmids
pUC19 (Yanisch-Perron et al., 1985)
pWUR397 (Brouns et al., 2008. cas3 under T7 promoter, KanR)
pWUR400 (Brouns et al., 2008. cascade genes under T7 promoter, StrR)
pWUR477 (Brouns et al., 2008. pACYCDuet-1 (Novagen) cloned with control spacers under T7 promoter, camR)
Enzymes
DpnI restriction enzyme (New England Biolabs, catalog number: R0176S )
T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S ) used with T4 DNA ligase buffer (New England Biolabs, catalog number: M0202S )
Kits
Gel and PCR Clean-up Kit (MACHEREY-NAGEL, catalog number: 740609.50 )
KAPA HiFi PCR Kit (Roche Diagnostics, catalog number: 07958935001 )
Lamda Taq PCR Master mix (Lamda Biotech, catalog number: D123P-200 )
Quick Ligation Kit (New England Biolabs, catalog number: M2200L )
Reagents
Agar (BD, DifcoTM, catalog number: 214010 )
Ampicillin (Merck, catalog number: 171254 ; Stock 100 mg/ml in double-distilled water, filtered, -20 °C)
Chloramphenicol (Merck, catalog number: 220551 ; Stock 35 mg/ml in ethanol, filtered, -20 °C)
Isopropyl-β-D-thiogalactopyranoside (IPTG) (Bio-Lab, catalog number: 16242352 ; Stock 1 M in double-distilled water, filtered, -20 °C)
Kanamycin (Merck, catalog number: 420311 ; Stock 50 mg/ml in 50% glycerol and double-distilled water, filtered, -20 °C)
L-arabinose (Gold Bio, catalog number: A-300-1 ; Stock 20% in double-distilled water, filtered, RT)
LB (Luria-Bertani) medium (10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl) (Acumedia)
Molecular biology water (Bio-Lab, catalog number: 232123 )
PEG 8000 (polyethylene glycol) (Promega, catalog number: V3011 ; Stock 50% in double-distilled water, filtered, RT)
Streptomycin (EMD Millipore, catalog number: 5711 ; Stock 50 mg/ml in double-distilled water, filtered, -20 °C)
TAE buffer (Bio-Lab, catalog number: 20502323 )
LB medium (see Recipes)
LB agar plates (see Recipes)
Soft agar (see Recipes)
TAE buffer (see Recipes)
Equipment
Cell density meter (OD600 reader) (Amersham Biosciences, model: Ultrospec 10 )
Electroporation device (Bio-Rad Laboratories, model: MicroPulserTM )
Gel electrophoresis apparatus (Cleaver Scientific, model: MultiSUB Choice )
Micro-centrifuge (Eppendorf, model: MiniSpin® )
NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000c )
Pipettes
Shaker (for 50 ml tubes, at 37 °C) (Thermo Fisher Scientific, Thermo ScientificTM, model: MaxQTM 2000 )
Thermal Cycler (Bio-Rad Laboratories, model: C1000 TouchTM )
Thermoblock (Labnet International, model: AccuBlockTM Digital Dry Baths )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Manor, M. and Qimron, U. (2017). Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System. Bio-protocol 7(15): e2431. DOI: 10.21769/BioProtoc.2431.
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Category
Microbiology > Microbial genetics > DNA
Molecular Biology > DNA > Mutagenesis
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2,432 | https://bio-protocol.org/exchange/protocoldetail?id=2432&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
Fuqiang Chen
XD Xiao Ding
YF Yongmei Feng
TS Timothy Seebeck
YJ Yanfang Jiang
GD Gregory D Davis
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2432 Views: 9462
Edited by: Jihyun Kim
Reviewed by: Vera Karolina Schoft
Original Research Article:
The authors used this protocol in 3-Apr 2017
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3-Apr 2017
Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of the chromatin, were also found to impede target DNA access and cleavage by SpCas9 in vitro (Knight et al., 2015; Hinz et al., 2015; Horlbeck et al., 2016; Isaac et al., 2016). Moreover, many CRISPR-Cas systems characterized to date often exhibit inactivity in mammalian cells and are thus precluded from gene editing applications even though they are active in bacteria or on purified DNA substrates. Thus, there is a need to devise a means to alleviate chromatin inhibition to increase gene editing efficiency, especially on difficult-to-access genomic sites, and to enable use of otherwise inactive CRISPR-Cas nucleases for gene editing need. Here we describe a proxy-CRISPR protocol for restoring nuclease activity of various class 2 CRISPR-Cas nucleases on otherwise inaccessible genomic sites in human cells via proximal targeting of a catalytically dead Cas9 (Chen et al., 2017). This protocol is exemplified here by using Campylobacter jejuni Cas9 (CjCas9) as nuclease and catalytically dead SpCas9 (SpdCas9) as proximal DNA binding protein to enable CjCas9 to cleave the target for gene editing using single stranded DNA oligo templates.
Keywords: CRISPR-Cas nuclease Cas9 dCas9 Cell culture Transfection Double strand breaks Gene editing
Background
By creating targeted chromosomal DNA double strand breaks (DSBs) or single strand breaks (nicks) or serving as a DNA binding module for other DNA modification effectors, programmable endonucleases have become an important tool for genome modification in eukaryotic cells (Gaj et al., 2013). In response to targeted DNA breaks, host cells can invoke various repair pathways to mend the damages to maintain the genome integrity. Insertions and/or deletions derived from NHEJ repair errors can be capitalized for gene knockout and homologous recombination can be exploited for introducing pre-determined changes on gene of interest by providing a DNA donor. In addition to these more traditional gene editing applications, catalytically inactive forms of programmable endonucleases are increasingly used as DNA binding modules for other DNA modification effectors, such as cytidine deamination enzymes (Komor et al., 2016). However, no matter which forms of programmable nucleases are utilized, target site binding is the prerequisite step and local chromatin structure can determine whether or not or how efficiently a programmable nuclease can bind the target site (Knight et al., 2015; Hinz et al., 2015; Horlbeck et al., 2016; Isaac et al., 2016, Chen et al., 2017). We hypothesize that binding at proximal locations by a programmable DNA binding protein could change the local chromatin structure and render an otherwise inaccessible target site accessible for binding.
Previous generations of programmable nucleases, such as meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), solely rely on protein structure to recognize target sites, and thus re-targeting of these nucleases requires rather laborious protein structural change. In contrast, class 2 CRISPR-Cas effector nucleases use protein structure to recognize a protospacer adjacent motif (PAM) and employ CRISPR RNA (crRNA) to bind the target site adjacent to the PAM. Because PAM is typically a short DNA sequence, such as 5’-NGG-3’ for SpCas9, and thus occurs frequently in a genome, re-targeting of CRISPR-Cas nucleases is a simple process of changing the crRNA sequence by molecular cloning or chemical synthesis. This targeting modality makes CRISPR-Cas systems very suitable for use as nucleases or as DNA binding proteins. This protocol combines these two utilities together to expand CRISPR gene editing capability. The CRISPR-Cas system used as nuclease must be orthogonal to the CRISPR-Cas system used as DNA binding protein to avoid binding site sharing. In general, different subtypes of class 2 CRISPR-Cas systems (e.g., type II-A, type II-B, type II-C, and type V) are orthogonal to one another. Within each subtype, some systems are highly divergent and could be also orthogonal to one another, but they need to be experimentally verified. Currently, it is highly recommended to use SpdCas9 as proximal DNA binding protein, for SpCas9 is the most robust system in mammalian cells to date, although it can also be inactive at certain genomic sites. However, it is anticipated that more robust Cas9 systems will be developed for use as DNA binding proteins.
Materials and Reagents
Pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2140 , 2149 , 2065E , 2069 , and 2079E )
75 cm2 U-shaped canted neck cell culture flask with vent cap (Corning, catalog number: 430641U )
Microcentrifuge tubes, 1.5 ml (Sigma-Aldrich, catalog number: T6649 )
Tissue culture plate, 6-well (Corning, Costar®, catalog number: 3516 )
Serological pipettes
5 ml (Corning, Costar®, catalog number: 4051 )
10 ml (Corning, Costar®, catalog number: 4488 )
25 ml (Corning, Costar®, catalog number: 4489 )
Cuvettes
Coverslip
50-ml conical centrifuge tube (Corning, catalog number: 430828 )
Human K562 cells (ATCC, catalog number: CCL-243 )
Catalytically inactive Streptococcus pyogenes Cas9 (SpdCas9) plasmid DNA (from MilliporeSigma; see Figure 1A)
SpCas9 sgRNA plasmid constructs with the guide sequences 5’-CCAAGGGTGAGGCCGGGAAG-3’ and 5’-CATCTCCCCCATGTACACCT-3’ for binding two human POR target sites (from MilliporeSigma; see Figure 1B)
Campylobacter jejuni Cas9 (CjCas9) plasmid DNA (from MilliporeSigma; see Figure 1C)
CjCas9 sgRNA plasmid construct with the guide sequence 5’-TTCGCCAGTACGAGCTTGTG-3’ for binding a human POR target site (from MilliporeSigma; see Figure 1D)
Figure 1. Vector maps
Single stranded DNA oligo donor for introducing a diagnostic EcoRI site at the CjCas9 cleavage site in POR. The oligo sequence is: 5’-CACCCTTGGTCTCCCCTTTCCAGCATTCGCCAGTACGAGCGAATTCTTGTGGTCCACACCGACATAGATGCGGCCAAGGTGTACATGG-3’ (Underlined: EcoRI site)
Note: Synthesize the oligo at 0.2 µmol scale and purify by PAGE. Re-suspend the oligo in 10 mM Tris buffer (pH 7.6) at 200 µM.
PCR primers for amplification of the targeted POR genomic region: forward 5’-CTCCCCTGCTTCTTGTCGTAT-3’, reverse 5’-ACAGGTCGTGGACACTCACA-3’
Alcohol
Hank’s balanced salt solution (Sigma-Aldrich, catalog number: H6648 )
Nucleofector instrument (Lonza, catalog number: AAB-1001 )
Amaxa Cell Line Nucleofector Kit V (Lonza, catalog number: VCA-1003 )
GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, catalog number: NA2010 )
JumpStart Taq ReadyMix (Sigma-Aldrich, catalog number: P2893 )
GenElute PCR Clean-Up Kit (Sigma-Aldrich, catalog number: NA1020 )
EcoRI restriction enzyme (New England Biolabs, catalog number: R0101S )
10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl (Bio-Rad Laboratories, catalog number: 4561036 )
Tris buffered saline (Sigma-Aldrich, catalog number: T5912 )
Gel loading buffer (Sigma-Aldrich, catalog number: G2526 )
DirectLoad Wide Range DNA Marker (Sigma-Aldrich, catalog number: D7058 )
Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, catalog number: I3390 )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
L-glutamine, 200 mM (Sigma-Aldrich, catalog number: G7513 )
K562 culture medium (see Recipes)
Equipment
Water bath (Polyscience)
Laminar flow hood for sterile tissue culture, biosafety level 2 approved (Thermo Fisher Scientific)
Microbiological incubator with 37 °C, atmospheric CO2 (Thermo Fisher Scientific)
Pipettes (Gilson)
Bench-top centrifuge (Eppendorf, model: 5417 C )
Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories, catalog number: 1658004 )
PCR thermocycler (Bio-Rad Laboratories, catalog number: 1861096 )
Software
ImageJ (https://imagej.nih.gov/ij/)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, F., Ding, X., Feng, Y., Seebeck, T., Jiang, Y. and Davis, G. D. (2017). Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting. Bio-protocol 7(15): e2432. DOI: 10.21769/BioProtoc.2432.
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Category
Molecular Biology > DNA > DNA modification
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2,433 | https://bio-protocol.org/exchange/protocoldetail?id=2433&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Liposome Disruption Assay to Examine Lytic Properties of Biomolecules
JJ John R. Jimah
PS Paul H. Schlesinger
NT Niraj H. Tolia
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2433 Views: 12792
Reviewed by: Venkatasalam Shanmugabalaji
Original Research Article:
The authors used this protocol in Dec 2016
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Original research article
The authors used this protocol in:
Dec 2016
Abstract
Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we describe an in vitro assay to evaluate the membrane lytic properties of proteins and biomolecules. Large unilamellar vesicles (liposomes) containing carboxyfluorescein at fluorescence-quenching concentrations are treated with the biomolecule of interest. A resulting increase in fluorescence due to leakage of the dye from liposomes and subsequent dilution in the buffer demonstrates that the biomolecule is sufficient for disrupting liposomes and membranes. Additionally, since liposome disruption may occur via pore-formation or via general solubilization of lipids similar to detergents, we provide a method to distinguish between these two mechanisms. Pore-formation can be identified and evaluated by examining the blockade of carboxyfluorescein release with dextran molecules that fit the pore. The methods described here were used to determine that the malaria vaccine candidate CelTOS and proapoptotic Bax disrupt liposomes by pore formation (Saito et al., 2000; Jimah et al., 2016). Since membrane lipid binding by a biomolecule precedes membrane disruption, we recommend the companion protocol: Jimah et al., 2017.
Keywords: Membrane Liposome Lipid Disruption Lysis Pore Liposome leakage Carboxyfluorescein Dextran
Background
This protocol presents the procedure to evaluate the membrane lytic properties of proteins and other biomolecules. This protocol aims to provide a clear description of the various experimental steps necessary to study the membrane disrupting properties of biomolecules. Finally, the protocol describes a quantitative measurement of membrane disruption and can be applied to provide insight into the kinetics and mechanism of liposome disruption. This protocol was successfully used to study the malaria vaccine candidate CelTOS that provided a clear description of the first in vitro functional assay for CelTOS. The application of this protocol revealed that CelTOS (cell traversal protein for ookinetes and sporozoites) disrupts membranes containing phosphatidic acid. This insight suggests that CelTOS is secreted by malaria parasites within invaded host cells to disrupt the host cell membrane and enable the exit of parasites (Jimah et al., 2016). Finally, this assay can be readily applied to investigate the inhibition of CelTOS-mediated liposome disruption by small molecules, antibodies or peptides. While designed for CelTOS, this protocol is readily generalizable and applicable to any other biomolecule of interest.
Materials and Reagents
Centrifuge tubes, 50 ml (Genesee Scientific, catalog number: 21-106 )
Microcentrifuge tubes, 1.7 ml (Midsci, catalog number: AVSS1700RA )
Parafilm (Bemis, catalog number: PM996 )
Glass Pasteur pipets (Fisher Scientific, catalog number: 13-678-20C )
Gravity sizing column (Bio-Rad Laboratories, catalog number: 7371032 )
Glass tubes with plain end (Fisher Scientific, catalog number: 14-961-27 )
Ring stand (Fisher Scientific, catalog number: S13747 )
Clamps (Fisher Scientific, catalog number: 02-217-005 )
Whatman filter (GE Healthcare, catalog number: 230300 )
200 nm polycarbonate Track-Etched filters (GE Healthcare, catalog number: 800281 )
Carboxyfluorescein (Sigma-Aldrich, catalog number: C0662 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
Sephadex G® G-25 (Sigma-Aldrich, catalog number: G25300 )
Phospholipids (dissolved in chloroform, as supplied by manufacturer). Commonly used phospholipids are:
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, catalog number: 850375C )
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457C )
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (Avanti Polar Lipids, catalog number: 840857C )
1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) (Avanti Polar Lipids, catalog numbers: 840034C )
Nitrogen gas tank (Airgas, catalog number: NI HP300 )
Triton-X 100 (Sigma-Aldrich, catalog number: X100 )
Dextran molecules of different molecular weights and radii of gyration:
5 kDa (Sigma-Aldrich, catalog number: 31417 )
9 kDa (Sigma-Aldrich, catalog number: D9260 )
39 kDa (Sigma-Aldrich, catalog number: D4133 )
Note: This product has been discontinued.
66.9 kDa (Sigma-Aldrich, catalog number: D1537 )
Note: This product has been discontinued.
148 kDa (Sigma-Aldrich, catalog number: D4876 )
500 kDa (Sigma-Aldrich, catalog number: D5251 )
Note: This product has been discontinued.
1,500-2,800 kDa (Sigma-Aldrich, catalog number: D5376 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Ethyl ether (Fisher Scientific, catalog number: E138-500 )
Ferric(III) chloride hexahydrate (Sigma-Aldrich, catalog number: 236489 )
Ammonium thiocyanate (Sigma-Aldrich, catalog number: 221988 )
Buffer-KCl (see Recipes)
Buffer-NaCl (see Recipes)
0.1 M ammonium ferrothiocyanate (see Recipes)
Equipment
Glass beaker (Fisher Scientific, catalog number: FB1012000 )
pH meter (Fisher Scientific, model: AccumetTM AE150, catalog number: 13-636-AE153 )
Stir bar
BenchRockerTM variable 2D rocker (Benchmark Scientific, catalog number: BR2000 )
Water sonicator (Laboratory Supplies, model: G112SP1T )
Gas pressure regulator (Linde, catalog number: UPE-3-75-580 )
Freeze-dry system for lyophilisation (Labconco, catalog number: 7750021 )
Hi-vac flask (Labconco, catalog number: 7544400 )
Vortexer (Fisher Scientific, catalog number: 02-215-414 )
Rotary flask evaporator: Rotovap (IKA, model: RV 8 FLEX , catalog number: 0010002178)
Round bottom flask, for rotovap (IKA, catalog number: 0003743200 )
Mini extruder (Avanti Polar Lipids, catalog number: 610023 )
Centrifuge (Beckman Coulter, model: J6-MI )
Cary Eclipse fluorescence spectrophotometer (Varian Inc./Agilent, Santa Clara, CA, USA)
UV/Vis/NIR spectrophotometer (capable of measuring optical density at 488 nm for Stewart assay) (Beckman Coulter, model: DU-640 )
Software
Microsoft Excel
GraphPad Prism
Procedure
Preparing 20 mM carboxyfluorescein
Weigh out appropriate mass of carboxyfluorescein to make 20 mM carboxyfluorescein in a desired volume.
Note: 40 ml is enough to make 80 preparations of liposomes. Each preparation is sufficient for 500 experimental assays.
Add ~70% of the target volume of buffer to the carboxyfluorescein powder.
Note: Commonly used buffers are buffer-KCl and buffer-NaCl (see the Recipes section). Identical buffers should also be used in all experiments, including all liposome and protein preparation steps.
While stirring the solution with a stir bar, titrate with 1 N KOH or NaOH a drop at a time.
Note: Be sure to not go above a pH of 8 as increasing the pH causes carboxyfluorescein to precipitate, and pH also affects the fluorescence spectra.
After the precipitate dissolves and the solution becomes dark, adjust the final pH to 7.4.
Note: The concentrated dark solution is fluorescence quenched.
Add appropriate volume of buffer to obtain a final concentration of 20 mM carboxyfluorescein.
Preparation of the size-exclusion chromatography column
Suspend 1 g of Sephadex® G-25 resin with 35 ml of buffer in a 50 ml conical tube.
Note: Commonly used buffers are buffer-KCl and buffer-NaCl.
Gently shake the tube at 30 rpm for 12-16 h at room temperature.
Add about 2 ml of buffer to the gravity column, and resin slurry in aliquots frequently enough to avoid layering in the packed column.
Note: The buffer provides resistance to the resin as it settles into the column and prevents the resin from forming channels.
During aliquot addition allow the column to flow slowly and continuously.
Note: Avoid introducing air bubbles when packing the column, and maintain buffer on top of the resin to prevent it from drying or forming channels.
Fill the ~30 ml gravity column about 95% to the top.
Note: Leave room at the top to add sample (liposomes). It is important to be able to see the top and bottom meniscus of dark orange colored liposomes through the clear glass stem of the sizing column.
The column can be reused indefinitely provided it has not dried out, the resin is not broken, and there are no air bubbles trapped in the resin.
Note: The column should be washed after each use, and equilibrated 3 times with 1 column volume of buffer before use.
Drying phospholipids
Test glass tubes for integrity by sonicating for 30 sec in a water sonicator.
Aliquot 10 mg of lipid (supplied by manufacturer with lipids dissolved in chloroform) into glass vial.
Clamp the vial to a stand while it is immersed in a beaker filled with water (Figure 1).
Figure 1. Drying phospholipids
Evaporate off all of the chloroform by flowing a nitrogen stream over the phospholipid solution until there is a thin layer of lipid.
Cover the test tube with Parafilm, and poke a small hole in the Parafilm.
Place the tube in a hi-vac flask, and lyophilize under vacuum for 3 h.
After drying the lipid, fill the vial with nitrogen and cover with Parafilm.
Note: Dried lipids can usually be stored for 1-3 months at -20 °C.
Making large unilamellar vesicles (liposomes) containing carboxyfluorescein
Add 500 µl of ethyl ether and 500 µl of the 20 mM carboxyfluorescein solution to the glass vial containing the dried lipid.
Cover the vial with Parafilm.
Note: Provide excess Parafilm because the Parafilm expands as the volatile gases escape from the vial.
Vortex for 5 sec to dissolve the lipids in the solution of ether and carboxyfluorescein.
Note: A layer of ether will sit on top of the green layer of carboxyfluorescein.
Sonicate the vial in a water sonicator three times for 30 sec each.
Note: Vortex sample for 15 sec between each sonication step. For optimal sonication, the vial should be at the center of the harmonic standing wave that forms in the water sonicator.
Use a Pasteur pipette to transfer the water-ether-phospholipid suspension into a round bottom flask.
Attach the round bottom flask to a rotary flask evaporator to remove the ether.
Note: Using a water pump vacuum start the vacuum low to avoid excess bubbling and to reduce bumping. Gradually increase the vacuum, for about a minute at a time as the ether is removed.
Release the vacuum and remove the round bottom flask from the roto-vap spindle. Sniff to determine if all the ether has evaporated. Usually, all the ether has evaporated 3 min after the sample stops bubbling while under the highest vacuum.
Notes:
Use the ‘wafting technique’ when sniffing.
At this step liposomes containing carboxyfluorescein will have typically formed as a suspension in the remaining buffer. However, it is critical to obtain liposomes of uniform size by extrusion.
Assemble the extruder (Avanti mini-extruder) with a 100 nm diameter cut off filter following the manufacturer’s directions (Figure 2).
Figure 2. Assembly of Avanti mini-extruder (Avanti Polar Lipids)
Notes:
Image from ‘Mini-Extruder Assembly Instructions’. Avanti Polar Lipids. Web. 19 June 2017 https://avantilipids.com/divisions/equipment/mini-extruder-assembly-instructions/.
All components above are included in the Avanti mini-extruder (Avanti Polar Lipids) except the ‘filter supports’ (Sigma-Aldrich) and ‘polycarbonate membrane’ (Sigma-Aldrich).
Pull buffer into one syringe and place it opposite to an empty syringe on the extrusion chamber. Gently inject the buffer across the extrusion chamber and membrane into the empty syringe.
Note: Injecting the buffer across the extruder wets and prepares it for liposomes. Empty out the receiving syringe, and place it back into the extruder.
Fill the injector syringe with the liposome suspensions and inject it through the extruder and passing it between the syringes five times. The liposome suspension will be at the receiving syringe. At this step, the solution contains liposomes that contain carboxyfluorescein and are of ~200 nm in diameter. However, the solution also contains a high concentration of carboxyfluorescein outside the liposomes, which needs to be removed.
Equilibrate the gravity sizing column with desired final buffer, of the same molarity as the buffer used for the liposome preparation.
Remove all of the buffer placing the meniscus on the top of the resin.
Gently add the liposome sample to the top of the resin, while causing minimal disruption of the packed resin.
Allow the column to flow, and stop it as soon as the entire liposome sample meniscus enters the resin.
Gently add buffer over the top of the beads to fill up the column. The dark orange/greenish colored liposome sample and the resin should remain intact (Figure 3A).
Allow the column to run.
Note: The liposomes, containing carboxyfluorescein, exceed the pore size of the resin and will elute in the void volume of the column. The free carboxyfluorescein molecules elute later because they enter the resin beads as they pass through the column (Figure 3B).
Collect the elution fractions containing liposomes, which are dark orange in color (Figure 1B).
Note: The free carboxyfluorescein is bright green and elutes after the liposomes. Expect about 0.5-1 ml of liposomes since liposomes were made by dissolving lipids in 500 µl of ether and carboxyfluorescein.
To determine the integrity of the liposomes, dilute the liposome fraction 1,000 fold and measure the fluorescence intensity at 512 nm upon excitation at 492 nm. Addition of 0.1% Triton-X 100 to the liposomes should result in approximately a threefold increase in fluorescence intensity.
Figure 3. Purifying liposomes containing carboxyfluorescein. A. Addition of sample to size-exclusion chromatography column. S: sample containing a mixture of liposomes containing carboxyfluorescein (L+CF), and free carboxyfluorescein outside liposomes (CF). B. Separation of liposome sample into two fractions. CF: free carboxyfluorescein that is bright green. L+CF: liposomes containing carboxyfluorescein that is orange in color.
Determining the lipid concentration
Note: If numerical studies are desired it is useful to determine the lipid content in the liposome preparation using the Stewart assay (Stewart, 1980). Lipid is lost during preparation especially during extrusion and gel filtration. The assay is based on the formation of a complex formed from the phospholipid and ammonium ferrothiocyanate, and sensitive for a lipid range of 0.01-0.1 mg.
To generate a standard curve, dissolve known amounts of lipids covering a range between 0.01 and 0.1 mg of lipid in chloroform to 2 ml. Five to ten points between 0.01 and 0.1 mg of lipid is sufficient to make a standard curve.
To determine the concentration of lipids in the prepared liposome sample, add an appropriate amount of chloroform to obtain a final volume of 2 ml.
Note: Since 10 mg of lipid was used to prepare liposomes, ensure that the sample is diluted enough to be in the range of 0.01 and 0.1 mg.
Add 2 ml of 0.1 M ammonium ferrothiocyanate (see Recipes) to each of the 2 ml volumes of chloroform containing known amounts of lipid and the test liposome sample.
Vortex the sample three times for 30 sec each, and centrifuge at 300 x g. With centrifugation, two layers will form.
Collect the lower layer with a Pasteur pipette.
Measure the optical density at 488 nm, and make a graph of the optical density versus lipid amount to obtain a standard curve.
The concentration of lipid in the liposome sample can be determined from the standard curve.
Preparing the biomolecule with potentially membrane lytic properties
Ensure that the biomolecule is dissolved in the same buffer as the liposomes.
Notes:
Commonly used buffers are buffer-KCl and buffer-NaCl. Remove detergent from the biomolecule or include detergent containing controls.
When testing a biomolecule for the first time, it is critical to use protein of good quality. Impurities, poor handling, and storage can lead to degradation, or aggregation of the protein which may impact the activity and result in the inaccurate quantification of protein activity. The type of purification needed to obtain homogenous protein samples, such as gravity column based affinity chromatography or gel filtration is dependent on each protein. It is recommended to use protein that elutes from a gel filtration column as a single monodisperse species. The purity of the protein can be determined by SDS-PAGE where the target protein is ~90% of the total protein sample.
Prepare a dilution series of the biomolecule. A range from low nanomolar to micromolar range can be recommended, since these are physiologically relevant concentrations for many proteins.
Liposome disruption assay (Figure 4)
Figure 4. Overview of the liposome disruption assay. The cuvette with buffer has no fluorescence. Addition of liposomes results in a minor increase in fluorescence due to residual free carboxyfluorescein outside the liposomes. Addition of lytic protein or biomolecule disrupts liposomes resulting in the release of carboxyfluorescein and subsequent dequenching of fluorescence. Finally, addition of Triton-X disrupts all the liposomes in the cuvette.
Set the spectrofluorimeter to record the fluorescence over time in order to determine the time dependence of liposome disruption. For carboxyfluorescein, program the fluorescence spectrophotometer to record the fluorescence emission at 512 nm upon excitation at 492 nm for a time range between 2 to 30 min.
Note: It is important to empirically determine the time range appropriate for each biomolecule because they may have different rates of liposome disruption.
Add 980 µl of buffer to a cuvette, and start the kinetic program to record the fluorescence emission over time.
Add 10 µl of liposomes from a stock to ensure a final desired lipid concentration (usually between 250 nM and 2.5 µM).
Note: Record the time that liposome sample is added. There should be a noticeable sudden increase in fluorescence mostly due to traces of free carboxyfluorescein outside liposomes remaining after previous purification steps.
Add 10 µl of the biomolecule or protein being tested.
Notes:
There will be a gradual increase in fluorescence, due to leakage of carboxyfluorescein from liposomes, if the protein disrupts liposomes.
Increase in fluorescence due to liposome disruption typically occurs within 5-30 min.
It is necessary to include a no protein control in order to determine the passive leakage of dye from the liposomes that is independent of a membrane lytic protein.
Finally, add 10 µl of 10% Triton-X to disrupt all liposomes in the system and obtain the 100% release value.
Dextran-block experiment to determine if liposome disruption occurs via pore formation.
Prepare 20 µM solutions of dextran molecules of different sizes dissolved in the buffer.
Note: A typical liposome disruption assay has a total lipid concentration of 250 nM to 1 µM. Meaning that using the Dextran (20 µM) is in excess of the liposomes in the experimental system.
Similar to the liposome disruption assay (described in Procedure G), determine the extent of liposome disruption in the absence of dextran in the buffer.
Determine the extent of liposome disruption with buffers containing different molecular weight dextran molecules. It is recommended to test dextran molecules ranging from 5 kDa to 2,000 kDa since the size of the pore is unknown.
Data analysis
Liposome disruption assay data analysis
The following formula is used to determine the percent of liposome disruption with time:
Disruptiontime = [(F512 ofliposome+protein - F512ofliposome)/(F512 ofliposome+triton - F512 ofliposome)] x 100%
Where F512 is the fluorescence intensity at 512 nm upon excitation at 492 nm.
For kinetic analyses, the time dependence of liposome disruption can be fitted to the following equation:
% LiposomesDisruptedtime = A[1 - e - (time/tau)] + m x time
where A is the maximum percentage of liposomes disrupted; Tau is the time constant for the exponential component and is the inverse of the rate constant (k); and m is the slope of the linear component.
Note: Fitting the data to this equation will provide data that can be used to obtain a hill plot as previously described (Saito et al., 2000). Additionally, examples of representative data and analysis are previously reported in Saito et al., 2000 and Jimah et al., 2016.
Dextran-block and pore-formation assay data analysis
A one-way ANOVA analysis using GraphPad can be used to determine if dextran molecules of a particular size block liposome disruption compared to the no dextran control.
This dextran-block experiment may reveal the size of the pore because radius of the pore will be similar to the radius of gyration of the dextran molecule that blocks liposome disruption.
Note: Examples of representative data and analysis are previously reported in Saito et al., 2000 and Jimah et al., 2016.
Notes
Good protein purification, handling, and storage are critical because membrane active proteins are frequently unstable and subject to aggregation, in addition the presence of proteases could degrade the protein resulting in a loss of activity. It is useful to determine the activity of freshly purified protein and determine how it compares to freeze-thawed samples.
Since proteins that disrupt membranes most likely have a solution form and membrane bound form, it might be necessary to activate the protein with detergent, or another molecule known to induce a conformational change in the protein. The amount of detergent or molecule should be enough to activate the protein without disrupting the liposomes and this must be tested with control samples.
Ensure that the liposomes are intact before testing the ability of protein to disrupt liposomes. Liposomes can be stored for approximately two days depending on the lipid composition. The fluorescence intensity of viable liposomes containing 20 mM carboxyfluorescein should increase three fold upon addition of 0.1% Triton-X.
The same buffers should be used in all steps to ensure there are no differences in osmolarity between the protein and liposome buffers. Differences in osmolarity may result in lysis of liposomes.
The Stewart assay may be less accurate because the absorbance reading of the chloroform phase, containing the lipid and ammonium ferrothiocyanate, may be skewed by the leakage or presence of carboxyfluorescein in the chloroform phase. An alternative 1H NMR based method for determining the phospholipid content of liposomes is described by Hein et al., 2016.
Exercise safety precautions when handling and storing solvents such as chloroform and ether.
Recipes
Buffer-KCl
10 mM HEPES pH 7.4
150 mM KCl
Buffer-NaCl
10 mM HEPES pH 7.4
150 mM NaCl
0.1 M ammonium ferrothiocyanate
Dissolve 27.03 g ferric chloride hexahydrate and 30.4 g ammonium thiocyanate in 1 L distilled water
Acknowledgments
This work was supported by the Burroughs Wellcome Fund (to NHT) and National Institutes of Health (R56 AI080792 to NHT). This protocol is adapted from Saito et al., 2000 and Jimah et al., 2016.
References
Hein, R., Uzundal, C. B. and Henning, A. (2016). Simple and rapid quantification of phospholipids for supramolecular membrane transport assays. Org Biomol Chem 4(7): 2182-5.
Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
Jimah, J. R., Schlesinger, H. P. and Tolia, H. N. (2017). Membrane lipid screen to identify molecular targets of biomolecules. Bio Protoc 7(15): e2427.
Saito, M., Korsmeyer, S. J. and Schlesinger, P. H. (2000). BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat Cell Biol 2(8): 553-555.
Stewart, J. C. (1980). Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 104(1): 10-14.
Copyright: Jimah et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Jimah, J. R., Schlesinger, P. H. and Tolia, N. H. (2017). Liposome Disruption Assay to Examine Lytic Properties of Biomolecules. Bio-protocol 7(15): e2433. DOI: 10.21769/BioProtoc.2433.
Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
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Category
Microbiology > Microbial biochemistry > Protein
Microbiology > Microbial biochemistry > Lipid
Biochemistry > Protein > Activity
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2,434 | https://bio-protocol.org/exchange/protocoldetail?id=2434&type=0 | # Bio-Protocol Content
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Observation of Pneumococcal Phase Variation in Colony Morphology
Jing Li
Juanjuan Wang
FJ Fangfang Jiao
Jing-Ren Zhang
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2434 Views: 8992
Reviewed by: Swetha Reddy
Original Research Article:
The authors used this protocol in Jul 2016
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Abstract
Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes pneumonia, meningitis, sepsis, and otitis media. This bacterium normally resides in the nasopharynx as a commensal, but sometimes disseminates to sterile sites of humans and causes local or systemic inflammation. This biphasic behavior of S. pneumoniae is correlated with a reversible switch between the opaque and transparent colony forms on agar plates, a phenomenon referred to as phase variation. The opaque variants appear to be more virulent in animal models of bacteremia but are deficient in nasopharyngeal colonization animal models. In contrast, the transparent variants display higher levels of nasopharyngeal colonization but relatively lower virulence in animal models. We have recently demonstrated that pneumococcal phase variation between these two colony types is caused by a reversible switch of genome DNA methylation (or epigenetic) patterns, which is driven by DNA inversions in the DNA methyltransferase genes. Observation of colony morphology is a simple and useful method to differentiate colonies with different characteristics, such as size, color, and opacity. This protocol describes how to study pneumococcal phase variation in colony morphology with a dissection microscope.
Keywords: Streptococcus pneumoniae Colony morphology Phase variation Epigenetic switch DNA inversion Dissection microscope
Background
Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children worldwide (Walker et al., 2013). The success of this pathogen in its adaptation to various ecological niches of human host depends on its remarkable phenotypic plasticity (Croucher et al., 2013; Johnston et al., 2014a), which has been reflected by the inter-strain antigenic variation in the capsular polysaccharides and surface proteins (Croucher et al., 2013 and 2011), acquisition of new virulence factors (Park et al., 2012), extensive drug resistance (Croucher et al., 2014) within the species. Natural genetic transformation is a well-known mechanism contributing to this phenotypic plasticity (Johnston et al., 2014b). Moreover, S. pneumoniae is also capable of spontaneous phase variation between opaque and transparent colony phenotypes, a widespread phenomenon in microbial pathogens (van der Woude, 2011). The opaque variants, which have more capsule and less teichoic acid, are more virulent in the lung and bloodstream; the transparent counterparts, which express less capsule and have more teichoic acid, are more adaptive to the nasopharynx (Kim and Weiser, 1998; Weiser et al., 1994; Manso et al., 2014; Li et al., 2016). Our recent studies have revealed that pneumococcal phase variation between two colony phenotypes is determined by DNA inversion between the methyltransferase hsdS genes in the colony opacity determinant (cod) locus (Feng et al., 2014; Li et al., 2016).
The protocol we present here describes the complete experimental procedure from preparing a bacterial stock to obtaining an image of colony morphology as described in our recent study (Li et al., 2016). Animal blood is commonly added to agar medium to promote growth of S. pneumoniae, but the color of the blood makes it difficult to differentiate opaque and transparent colonies by microscopic approach. Instead, catalase is supplemented to agar plates to culture S. pneumoniae by neutralizing the inhibition effect of hydrogen peroxide (produced by the pneumococci themselves) on pneumococcal growth when it comes to observe and document colony morphologies by dissection microscope (Kim and Weiser, 1998; Weiser et al., 1994; Manso et al., 2014; Li et al., 2016). Since colony morphology phenotypes can be indicative of bacterial physiological and pathogenic properties, this protocol may offer a valuable method to study the impact of genetic and epigenetic elements or environmental conditions on bacterial biology and disease pathogenesis.
Materials and Reagents
Pipette tips
Petri dishes (100 mm) (Thermo Fisher Scientific, Thermo Scientific TM, catalog number: 263991 )
50 ml tubes (Thermo Fisher Scientific, Thermo Scientific TM, catalog number: 339652 )
0.2 μm filter (Pall, catalog number: 4612 )
1.5 ml tubes (Eppendorf, catalog number: 022363204 )
Spreading rods (Sigma-Aldrich, catalog number: Z376779 )
S. pneumoniae strain ST556 (Li et al., 2012)
Todd Hewitt broth (Sigma-Aldrich, catalog number: T1438 )
Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Tryptic soy agar (TSA) (BD, DifcoTM, catalog number: 236950 )
Catalase from bovine liver (Sigma-Aldrich, catalog number: C9322 )
Phosphate buffered saline (PBS) (Mediatech, catalog number: 21-040-CV )
Todd Hewitt broth with yeast extract (THY) medium (see Recipes)
TSA medium (see Recipes)
Catalase working solution (see Recipes)
Equipment
Pipettes
Dissection microscope (ZEISS, model: Stemi 2000-C )
37 °C water bath
Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: GENESYSTM 30 )
37 °C 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
Autoclave (SANYO, model: MLS-3780 )
Analytical balance (Mettler-Toledo International, model: NewClassic ML802 )
Vortex mixer
Centrifuge (Eppendorf, model: 5417 R )
Class II biological safety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM Class II )
Digital single lens reflex camera (Canon, model: EOS 550D )
Small mirror with a diameter of ~5 cm
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Li, J., Wang, J., Jiao, F. and Zhang, J. (2017). Observation of Pneumococcal Phase Variation in Colony Morphology. Bio-protocol 7(15): e2434. DOI: 10.21769/BioProtoc.2434.
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Category
Microbiology > Microbe-host interactions > Bacterium
Microbiology > Microbial biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Polysaccharide
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2,435 | https://bio-protocol.org/exchange/protocoldetail?id=2435&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells
YZ Yong-Wei Zhang
RN Rochelle E. Nasto
SJ Sandra A. Jablonski
IS Ilya G Serebriiskii
RS Rishi Surana
JM Joseph Murray
MJ Michael Johnson
RR Rebecca B. Riggins
RC Robert Clarke
EG Erica A. Golemis
LW Louis M. Weiner
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2435 Views: 8517
Reviewed by: Aswad KhadilkarVarpu Marjomaki
Original Research Article:
The authors used this protocol in Mar 2016
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Abstract
RNAi screening technology has revealed unknown determinants of various biological signaling pathways in biomedical studies. This protocol provided detailed information about how to use RNAi screening to identify proliferation determinants in breast tumor cells. siRNA-based libraries targeting against Estrogen receptor (ER)-network, including 631 genes relevant to estrogen signaling, was constructed for screening in breast cancer cells. Briefly, reverse transfection of siRNA induced transient gene knockdown in MCF7 cells. First, the transfection reagent for MCF7 cells was selected. Next, the Z’-score assay was used to monitor if screening conditions yielded efficiently. Then, the ER-network siRNA library screening was preceded by automatic machines under optimized experimental conditions.
Keywords: RNA interference (RNAi) Screening Estrogen receptor (ER) Breast cancer Z’-score Multidrop Combi-nL reagent dispenser WellMate microplate dispenser CyBio automatic dispenser
Background
RNA interference (RNAi) is a biological process that can be exploited to inhibit gene expression by causing the destruction of specific mRNA molecules. Knockdown of specific genes by RNAi technology is often associated with phenotypic changes, which has made RNAi widely used in life science research. Two systems are utilized for high-throughput RNAi screening, one is lentivus-based short hairpin RNA (shRNA) library screening; the other is chemical synthesized small interference RNA (siRNA)-based screening (Boutros et al., 2008). shRNA-based transfection induces stable gene knockdown in cells. siRNA-based transfection induces transient gene knockdown. Lentiviral pooled shRNA libraries contain lentiviruses with shRNAs targeting against either genomic DNA or a group of genes. Following analysis is required to distinguish target genes after screening, such as chip-based DNA microarray or next generation sequencing (NGS). However, in siRNA-based libraries, siRNAs against each single target gene are distributed in each well of 96-well or 384-well plates. A siRNA library may include many plates depending on the number of targeting genes in this library. For siRNA library screening, no further techniques are required to identify targeting genes.
In our studies, we designed the Estrogen receptor (ER)-network around 5 seed proteins relevant to estrogen signaling: the ER genes ESR1 (ERα) and ESR2 (ERβ), the estrogen-related receptors ESRRA and ESRRG, and CYP19A1 (aromatase). 631 genes were selected as ER network. Next, we constructed siRNA-based libraries targeting against ER network genes into 96-well plates, which were custom-made from QIAGEN (MD, USA). siRNAs against those genes were distributed into 11 x 96-well plates. Two siRNAs were selected for each gene and mixed in one well (Zhang et al., 2016). The advantage of our method provides high-throughput screening by using automatic machines (Cybio, Combi-nL or Wellmate dispenser) to dispense liquid to speed the screening process.
Different types of cancer cell lines had been used in RNAi screening with our methods (Astsaturov et al., 2010; Murray et al., 2014; Zhang et al., 2016), such as estrogen positive breast cancer MCF7, estrogen-independent MCF7 (LCC1 and LCC9), triple negative breast cancer MDA-MB-231, epidermoid cancer A431 and human fibroblast HFF1 cells etc. For each cell line, the optimal transfection reagent has to be determined before RNAi library screening. Z’-score is taken as a quantitative parameter to control the experiment quality for various cell lines and corresponding transfection reagents. In this assay, we utilize ER-network RNAi screening in MCF7 cells as an example to describe the protocol (Zhang et al., 2016). It also fits other cell lines or other gene network RNAi library with minor modification, such as type of transfection reagent, cell plating density, Cell Titer blue incubation time or RNAi library scale (total number of siRNA library plates), which will be noted. In this article, these protocols will be described in three parts: 1) Selection of transfection reagents; 2) Z’-score determination; 3) Screening an RNAi library.
Materials and Reagents
Pipette tips for CyBi-Well Vario 96 channel simultaneous Pipettor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5587 )
V-bottom 96-well plates (Corning, catalog number: 3357 )
Flat-bottom 96-well plates (Corning, catalog number: 3595 )
50 ml conical tube
Corning 0.22 µm vacuum filter system (Corning, catalog number: 431098 )
T75 flasks (Corning, Costar)
Labels with Barcode
MCF7 cells (Tissue Culture Shared Resource, Lombardi Cancer Center, Georgetown Univ.)
AllStars Negative Control siRNA (QIAGEN, catalog number: 1027281 )
AllStars Hs Cell Death siRNA (QIAGEN, catalog number: 1027299 )
AP2A siRNA (QIAGEN, catalog number: SI04371283 )
GRB14 siRNA (QIAGEN, catalog number: SI00430703 )
Opti-MEM reduced serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
IMEM medium (Mediatech, catalog number: 10-024-CV )
Trypsin-EDTA (0.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15400054 )
Charcoal-stripped bovine calf serum (CCS) (Gemini Bio-Products, catalog number: 100-213 )
Estradiol (Sigma-Aldrich, catalog number: E8875 )
Cell Titer Blue (Promega, catalog number: G8082 )
Hank’s balanced salt solution (HBSS) without calcium, magnesium, phenol red (GE Healthcare, HycloneTM, catalog number: SH30588.01 )
ER network siRNA library plates (Customized from QIAGEN)
siRNA suspension buffer (QIAGEN)
Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778500 )
HiPerfect (QIAGEN, catalog number: 301704 )
Dharmafect 1-4 transfection reagent (GE Dharmacon, catalog numbers: T-2001 , T-2002 , T-2003 , T-2004 )
RNAiFect (QIAGEN)
70% (v/v) ethanol (filtered via Corning 0.22 µm vacuum filter system)
0.22 µm filtered ddH2O
Equipment
CyBi-Well Vario 96 channel simultaneous Pipettor (CyBio)
Multidrop Combi-nL reagent dispenser (Thermo Fisher Scientific, catalog number: 5840400 )
WellMate microplate dispenser (Thermo Scientific Matrix)
AccuSpin 3R Centrifuge with Ch.003741 rotor (Thermo Fisher Scientific, catalog number: 4393 ) and swing rectangular buckets with adapters (Thermo Fisher Scientific, catalog number: 75006449 )
Magnetic stirrer (Thermo Fisher Scientific)
Envision multi-label plate reader with 560Ex/590Em filter set (PerkinElmer, catalog number: 2104-0010 )
500 ml glass bottle (Corning, Costar)
Part I. Selection of transfection reagents
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhang, Y., Nasto, R. E., Jablonski, S. A., Serebriiskii, I. G., Surana, R., Murray, J., Johnson, M., Riggins, R. B., Clarke, R., Golemis, E. A. and Weiner, L. M. (2017). RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells. Bio-protocol 7(15): e2435. DOI: 10.21769/BioProtoc.2435.
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Category
Cancer Biology > General technique > Cell biology assays
Cell Biology > Cell-based analysis > Gene expression
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2,436 | https://bio-protocol.org/exchange/protocoldetail?id=2436&type=0 | # Bio-Protocol Content
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Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings
Yonghui Zhao
Shengben Li
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2436 Views: 11476
Edited by: Arsalan Daudi
Reviewed by: Liping ShenWenrong He
Original Research Article:
The authors used this protocol in Dec 2016
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Dec 2016
Abstract
The plant endomembrane system plays vital roles for synthesis, modification and secretion of proteins and lipids. From the classic view, only mRNAs encoding secreted proteins could be targeted to the endoplasmic reticulum (ER) for translation via a co-translational translocation manner, however, recently this model has been challenged by accumulative evidence that lots of cytosolic mRNAs could also associate with ER, and that some categories of small RNAs are enriched on ER. These results suggested unrevealed functions of ER beyond our current knowledge. The large scale identification of RNAs and proteins on microsome is crucial to demonstrating the ER function and the studies will be boosted by next generation sequencing technology. This protocol provides a technical workflow to isolate the cytosol, microsome, free polysome (FP) and membrane bound polysome (MBP) from plant tissue. The isolated fractions are suitable for genome wide profiling of mRNAs, small RNAs and proteins.
Keywords: Cytosol Microsome Free polysome Microsome bound polysome
Background
Plant endomembrane system is very important for cell wall formation, lipid biosynthesis, protein synthesis, modification, folding and trafficking. According to the co-translational translocation model, signal peptides at the N-terminal of secreted proteins are synthesized by cytosolic polysomes, and then recognized by signal recognition particles on ER, and the remaining portion of proteins will be subsequently synthesized on ER. According to this model, only mRNAs encoding for secreted proteins could be brought to ER for translation (Peter and Johnson, 1994). However, large portion of mRNAs were identified from mammalian and plant cell ERs (Lerner et al., 2003; de Jong et al., 2006), and recent studies revealed that ER also functions as a key hub for small RNA function in plant (Li et al., 2013 and 2016). These findings broadened our knowledge about ER functionality. Large scale identification of mRNAs, small RNAs and proteins from ER of cells upon different developmental stages and environmental stimuli will provide valuable clues for elucidating new functions of ER. Here, we describe a protocol to isolate the cytosol, microsome, FP and MBP from Arabidopsis thaliana, and it could be adapted to rice, maize and other plants.
Materials and Reagents
Pipette tip (Denville Scientific, catalog numbers: P2101 , P2102 , P2109 ), autoclave before use
50 ml tube
Miracloth (EMD Millipore, catalog number: 475855-1R )
15 ml tube
13 x 51 mm centrifuge tubes (Beckman Coulter, catalog number: 326819 )
25 x 89 mm centrifuge tubes (Beckman Coulter, catalog number: 355631 )
Arabidopsis ecotype Columbia-0 maintained by our own laboratory
Murashige and Skoog medium
Liquid nitrogen
1% (v/v) Triton X-100
DEPC H2O
Tris base (Fisher Scientific, catalog number: BP152-5 )
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A142-212 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
MgOAc
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
Sucrose (Fisher Scientific, catalog number: BP220-212 )
Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
Cycloheximide (Sigma-Aldrich, catalog number: C1988 )
Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
Ethanol
SUPERaseIN (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2696 )
Magnesium acetate tetrahydrate (MgCl2·4H2O) (Sigma-Aldrich, catalog number: M5661 )
Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: BP214-500 )
Proteinase inhibitor cocktail-EDTA free (Roche Diagnostics, catalog number: 18970600 )
Ribosome extraction buffer (see Recipes)
Sucrose cushion buffer (see Recipes)
Resuspending buffers (see Recipes)
10x sucrose salt (for 15-60% sucrose gradient column) (see Recipes and Notes)
Equipment
Pipette (Eppendorf)
Plant growth chamber (Percival Scientific, model: CU-36L4 )
L8-70M Ultracentrifuge (Beckman Coulter, model: L8-70M )
SW 28 rotor (Beckman Coulter, model: SW 28 Ti )
SW 55 Ti rotor (Beckman Coulter, model: SW 55 Ti )
Type70 Ti rotor (Beckman Coulter, model: Type70 Ti )
25 x 89 mm bottle, with cap assembly (Beckman Coulter, catalog number: 355618 )
Vacuum pump
Centrifuge (Eppendorf, model: 5424 R )
High speed centrifuge (Beckman Coulter, model: Avanti J-E Series )
NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
Density gradient fractionation systems (BRANDEL, model: BR-188 )
37 °C incubator
Software
Data acquisition software (Brandel, model: PEAK CHART)
Procedure
This protocol allows the simultaneous isolation of FP and MBP from the same plant sample. Briefly, the cytosol and microsome fractions are separated by centrifugation, and microsome fraction is dissolved with extraction buffer supplemented with detergent. Both cytosol and microsome lysates are passed through sucrose cushion solution by ultracentrifugation to obtain FP and MBP pellets which are subsequently subjected to density gradient fractionation and profile analyses (Figure 1).
Figure 1. Scheme of FP and MBP isolation
Col-0 seeds are sterilized and plated on Murashige and Skoog medium, and plants are grown in a growth chamber at 23 °C under 16 h light/8 h dark cycles for 12 days.
2 g seedlings are ground into fine powder in liquid nitrogen, and are suspended in 8 ml ribosome extraction buffer (see Recipes) in a 50 ml tube. Keep on ice for 20 min.
The slurry is filtered with 2 layers of Miracloth to a 15 ml tube and centrifuged twice at 10,000 x g for 10 min to remove the debris.
The supernatant is transferred into a Beckman centrifuge tube and centrifuged at 30,000 x g for 30 min with a Beckman SW28 rotor. Transfer the supernatant to a new tube as the cytosol fraction and keep it on ice.
Resuspend the pellet with 8 ml ribosome extraction buffer followed by centrifugation at 30,000 x g for another 30 min. Discard the supernatant, and the pellet is kept as the microsome fraction.
Note: The cytosol and microsome fractions from steps 3 and 4 are ready for RNA and protein extraction. If you want to perform FP/MBP isolation, please continue the following steps.
Dissolve the microsome pellet with 8 ml ribosome extraction buffer supplemented with 1% (v/v) Triton X-100. Keep it on ice for 20 min.
Subject the cytosol extract (step 3) and microsome lysate (step 5) to centrifugation at 30,000 x g for 30 min with a Beckman SW28 rotor to remove any residual membranes.
Transfer 8 ml sucrose cushion solution (see Recipes) into a centrifugation bottle (Beckman centrifuge) suitable for Type70 Ti rotor (Beckman Coulter), and then slowly load the clarified cytosol or microsome lysate from step 6 on the top of the sucrose cushion.
Note: Be careful not to disturb the sucrose cushion layer.
Centrifuge at 183,960 x g with Type70 Ti rotor (Beckman Coulter) at 4 °C for 3 h.
Draw a circle around the ribosome pellet with a marker pen, and remove all liquid in the tube with a pipette or a vacuum pump (Video 1). Hold the tube with the marked position upward, and carefully wash the inner surface of the tube except for the marked area by 1 ml ddH2O three times with a pipette (Video 1). The purpose of this step is to remove the residual salt and sucrose in the tube. Any touching with the FP/MBP pellet either by pipette tip or water must be avoided.
Video 1. Removal of supernatant by vacuum and washing of the tube inner wall
Resuspend the pellets in 400 μl resuspension buffer (see Recipes), and transfer them to nuclease free microcentrifuge tubes. Keep the tubes on ice for 30 min.
Centrifuge at 16,000 x g for 5 min at 4 °C to remove debris, and transfer the supernatants to new tubes.
Note: The samples obtained from step 11 are ready for RNA and protein extraction of FP/MBP. If you want to check the FP/MBP profiles, continue the following steps.
Measure the OD260 of the samples from step 11 with NanoDrop spectrophotometer.
Slowly load 1,000 OD260 of FP or 200 OD260 of MBP on the top of 15-60% sucrose gradient column (see Notes). The yield of MBP is much lower than FP, but 200 OD260 is enough for the MBP profile analysis.
Note: Be careful not to disturb the sucrose gradient. It is important to keep the pipette tip and the surface of the gradient solution nicely touched (but not protruding into the solution) during the loading, otherwise droplets may be formed and the gradient will be disturbed.
Centrifuge at 237,020 x g with SW55 Ti rotor (Beckman Coulter) for 1.5 h at 4 °C.
Perform the density gradient fractionation. The fractionation system is composed of a syringe pump, a tube piercer stand, a detector, a fraction collector and the Peakchart software (Figure 2A). The gradient column is mounted onto the tube piercer stand and is pierced by the needle at the bottom of stand (see Video 2). For the syringe pump, put the speed mode switch to ‘normal’ position, and the fluid direction switch to ‘off’ position; Turn the control mode knob to ‘remote start/stop’, and adjust the fluid speed to 1.5 ml/min (Figure 2B); For the UA-6 detector, set the sensitivity value as ‘1’, and the chart speed as ‘150 cm/h’ (Figure 2C). The system was under control of the Peakchart software, and ribosome profiles were recorded by the software and the detector (Figure 2D).
Figure 2. The density gradient fractionation system. A. The overview of the fractionation system. The gradient column is attached to the tube piercer stand, and pierced by the needle at the bottom of the stand. The gradient solution is slowly pushed out from the top of the column by the chase fluid in the syringe pump, and A254 nm absorbance was recorded by the UA-6 detector and the Peakchart software. B. The front panel of the syringe pump. The positions of the switches and knobs reflect the parameter settings during fractionation. C. The front panel of the UA-6 detector. The positions of the knobs reflect the parameter settings during fractionation. D. A screenshot of the Peakchart software. The start or stop of the entire system is controlled by the green button at the bottom center, the profile is shown on the screen in real time manner, and the data are automatically saved when the procedure completes.
Video 2. Attachment of the gradient column to the density gradient fractionation system
Analyze the ribosome profile. The typical FP/MBP profiles are shown in Figure 3. The different peaks represent 40S small subunit, 60S large subunit, the 80S monoribosome and polyribosomes respectively, and a good isolation of FP or MBP should display a profile with these distinct peaks and a hill shaped pattern instead of a decline curve in the polysome region. Note that the peak of 80S monomer of MBP is much lower than that of FP, and 60S and 80S fractions were usually combined in MBP.
Figure 3. Profiles of FP and MBP. FP (A) and MBP (B) are separated in 15-60% sucrose gradient by ultracentrifugation, and are fractionated by gradient fractionation system subsequently. The x-axis indicates the sucrose concentration in the corresponding gradient, and the y-axis represents the absorbance level at 254 nm. 40S: small subunit of ribosome; 60S: large subunit of ribosome; 80S: the monoribosome complex.
Notes
Preparation of 15-60% sucrose gradient column. All stock solutions are prepared with DEPC H2O except for CHX and CHL which were prepared with ethanol.
Prepare sucrose solutions with different sucrose concentration (for 10 gradient columns, Table 1):
Table 1. The recipes for preparing the 15-60% sucrose gradient column
Place 13 x 51 mm centrifuge tubes (Beckman) into a rack that can withstand -80 °C
Start with the 60% sucrose layer, pipette 0.75 ml 60% sucrose solution into a 13 x 51 mm centrifuge tube (Beckman), avoiding any air bubbles, and then freeze for 1h at -80 °C.
Add the next gradient layer with the volumes indicated in the table, freeze again, and continue with the last two layers.
Store the sucrose gradient columns at -80 °C. The gradient columns could be used in 3 months if they are stored properly.
Before use, remove the column from the freezer, and thaw in a 37 °C incubator for exactly 1 h followed by cooling down in a cold room or refrigerator for another 1 h.
Recipes
Ribosome extraction buffer
0.2 M Tris-HCl, pH 8.5
0.1 M KCI
70 mM MgOAc
50 mM EGTA
0.25 M sucrose
10 mM DTT
50 μg/ml Cycloheximide (CHX) (stock 50 μg/μl in ethanol)
50 μg/ml Chloramphenicol (CHL) (stock 50 μg/μl in ethanol)
2.5 U/ml SUPERaseIN
Sucrose cushion solution
0.4 M Tris-HCl, pH 9.0
0.2 M KCI
0.005 M EGTA
0.035 M MgCl2
1.75 M sucrose
5 mM DTT
50 μg/ml CHX (stock 50 μg/μl in ethanol)
50 μg/ml CHL (stock 50 μg/μl in ethanol)
Resuspension buffer
0.2 M Tris-HCl, pH 9.0
0.2 M KCl
0.025 M EGTA
0.035 M MgCl2
5 mM DTT
50 μg/ml CHX (stock 50 μg/μl in ethanol)
50 μg/ml CHL (stock 50 μg/μl in ethanol)
10x sucrose salt (for 15-60% sucrose gradient column)
0.4 M Tris-HCl, pH 8.4
0.2 M KCI
0.1 M MgCI2
Note: All buffers were prepared with DEPC water if not emphasized; DTT, CHX, CHL and SUPERaseIN need be added freshly.
Acknowledgments
This protocol was adapted from our previous work (Li et al., 2016). We thank Dr. Xuemei Chen for suggestions on this protocol. The work was supported by grants from the science technology and innovation committee of Shenzhen municipality (JCYJ20151116155209176, KQCX2015033110464302, KY20150114), and the Key Laboratory of Shenzhen (ZDSYS20141118170111640).
References
de Jong, M., van Breukelen, B., Wittink, F. R., Menke, F. L., Weisbeek, P. J. and Van den Ackerveken, G. (2006). Membrane-associated transcripts in Arabidopsis; their isolation and characterization by DNA microarray analysis and bioinformatics. Plant J 46(4): 708-721.
Lerner, R. S., Seiser, R. M., Zheng, T., Lager, P. J., Reedy, M. C., Keene, J. D. and Nicchitta, C. V. (2003). Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes. RNA 9(9): 1123-1137.
Li, S., Le, B., Ma, X., Li, S., You, C., Yu, Y., Zhang, B., Liu, L., Gao, L., Shi, T., Zhao, Y., Mo, B., Cao, X. and Chen, X. (2016). Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis. Elife 5.
Li, S., Liu, L., Zhuang, X., Yu, Y., Liu, X., Cui, X., Ji, L., Pan, Z., Cao, X., Mo, B., Zhang, F., Raikhel, N., Jiang, L. and Chen, X. (2013). MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153(3): 562-574.
Peter, W. and Johnson, A. E. (1994). Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol 10: 87-119.
Copyright: Zhao and Li. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Zhao, Y. and Li, S. (2017). Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings. Bio-protocol 7(15): e2436. DOI: 10.21769/BioProtoc.2436.
Li, S., Le, B., Ma, X., Li, S., You, C., Yu, Y., Zhang, B., Liu, L., Gao, L., Shi, T., Zhao, Y., Mo, B., Cao, X. and Chen, X. (2016). Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis. Elife 5.
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Plant Science > Plant cell biology > Organelle isolation
Cell Biology > Organelle isolation > Microsome
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2,437 | https://bio-protocol.org/exchange/protocoldetail?id=2437&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans
Tabea Dierker
LK Lena Kjellén
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2437 Views: 11235
Edited by: Peichuan Zhang
Original Research Article:
The authors used this protocol in Oct 2016
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Oct 2016
Abstract
The nematode Caenorhabditis elegans is a popular model organism for studies of developmental biology, neurology, ageing and other fields of basic research. Because many developmental processes are regulated by glycosaminoglyans (GAGs) on cell surfaces and in the extracellular matrix, methods to isolate and analyze C. elegans GAGs are needed. Such methods have previously been optimized for other species such as mice and zebrafish. After modifying existing purification protocols, we could recently show that the nematodes also produce chondroitin sulfate, in addition to heparan sulfate, thus challenging the view that only non-sulfated chondroitin was synthesized by C. elegans. We here present our protocol adapted for C. elegans. Since the purification strategy involves separation of non-sulfated and sulfated GAGs, it may also be useful for other applications where this approach could be advantageous.
Keywords: Glycosaminoglycans Caenorhabditis elegans Proteoglycans Ion exchange chromatography Sulfation
Background
Glycosaminoglycans (GAGs) are linear polysaccharide chains of repeating disaccharide units, which are often substituted with sulfate groups. Except for hyaluronan, which is a non-sulfated GAG synthesized at the plasma membrane without being anchored to any protein, all other GAGs are covalently linked to core proteins, thus forming proteoglycans (PGs). The most common GAGs found on PGs are heparan sulfate (HS) and chondroitin sulfate (CS)/dermatan sulfate, containing N-acetyl-glucosamine and N-acetyl-galactosamine, respectively (Zhang, 2010). During their biosynthesis in the Golgi compartment, which is a non-template driven process, they are subject to multiple modifications, including epimerization of glucuronic acid into iduronic acid and sulfation at various positions (Bulow and Hobert, 2006; Zhang, 2010). The sulfation patterns generated are important for the ability of the GAG chains to interact with growth factors and cytokines, which in turn is essential for their ability to influence development and other important physiological processes (Bishop et al., 2007).
In order to analyze their composition, GAGs need to be purified from crude cell or tissue lysates, most commonly achieved by ion exchange chromatography after protease and nuclease digestion (Ledin et al., 2004). Disaccharides, generated by the action of specific GAG lyases, can then be identified using different chromatography methods or mass spectrometry (Shao et al., 2013; Kiselova et al., 2014). We and several other labs have used reversed-phase ion-pairing (RPIP)-HPLC for detection of different types of GAGs in multiple species (Ledin et al., 2004; Lawrence et al., 2008; Yamada et al., 2011; Holmborn et al., 2012).
C. elegans synthesizes HS with modifications similar to those found in mammals and other organisms, but like other ecdysozoa the nematodes do not produce hyaluronan (Yamada et al., 1999; Toyoda et al., 2000; Lawrence et al., 2008; Townley and Bulow, 2011). However, although vast amounts of non-sulfated chondroitin were detected, CS was not previously identified, giving C. elegans a unique position among the ecdysozoa (Yamada et al., 1999; Toyoda et al., 2000).
In our protocol we separated sulfated and non-sulfated GAGs prior to analysis, facilitating detection of CS occurring in much lower quantities than the non-sulfated chondroitin (Dierker et al., 2016). Here, this method is described in detail.
Materials and Reagents
Pipette tips (SARSTEDT, catalog numbers: 70.1114.100 , 70.760.502 , 70.762.200 )
Tissue paper
15 ml tubes (SARSTEDT, catalog number: 62.554.502 )
50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
1.5 ml Screwlock tubes (SARSTEDT, catalog number: 72.692.100 )
Syringes 1 ml (Terumo Medical, catalog number: SS+01T1 )
Syringes 2 ml (BD, catalog number: 300185 )
MicrolanceTM 3 18 G needles (VWR, catalog number: 613-3945 )
MicrolanceTM 3 23 G needles (VWR, catalog number: 613-3923 )
MicrolanceTM 3 27 G needles (VWR, catalog number: 613-3834 )
10 ml round-bottom tubes
Parafilm
0.45 µm filters (for example EMD Millipore, catalog number: HAWP04700 )
Petri dishes 10 cm (SARSTEDT, catalog number: 82.1473.001 )
10 ml Poly-Prep® Chromatography columns (Bio-Rad Laboratories, catalog number: 7311550 )
DEAE Sephacel (GE Healthcare, catalog number: 17050001 )
1.5 ml reaction tubes (SARSTEDT, catalog number: 72.690.001 )
2 ml reaction tubes (SARSTEDT, catalog number: 72.695.500 )
NAP-10 columns (GE Healthcare, catalog number: 17-0854-02 )
HPLC tubes (VWR, catalog number: 548-0440 )
Locks (Fisher Scientific, catalog number: 11521434 )
Note: This product has been discontinued.
C. elegans and bacterial strains
Note: All C. elegans strains as well as E. coli strain HB101 were obtained from the Caenorhabditis Genetics Center (CGC) https://cbs.umn.edu/cgc/home.
Sodium hypochlorite (NaClO 6-14% active Cl) (Sigma-Aldrich, catalog number: 13440 )
Protease XIV (Sigma-Aldrich, catalog number: P5147-5G )
Benzonase (Merck, catalog number: 70746-3 )
Heparin lyase I, II, III (IBEX Pharmaceuticals, catalog numbers: 50-010 , 50-011 , 50-012 )
Quant-iTTM Broad Range Assay Kit (purchased from Molecular Probes)
Calcium chloride (CaCl2•2H2O) (Merck, catalog number: 1.02382.0500 )
Potassium hypochlorite (KOH) (Sigma-Aldrich, catalog number: P1767 )
Potassium hydroxide (KOH) (Merck, catalog number: 1.05021.0250 )
Magnesium sulfate heptahydrate (MgSO4•7H2O) (Merck, catalog number: 1.05886 )
Cholesterol (Sigma-Aldrich, catalog number: C8667 )
Ethanol (SOLVECO, catalog number: 1015 )
Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 1.04873.1000 )
di-Potassium hydrogen phosphate trihydrate (K2HPO4•3H2O) (Merck, catalog number: A257899 )
Note: This product has been discontinued.
1 M K2HPO4
Sodium chloride (NaCl) (Riedel-de Haën, catalog number: 31439 )
Agarose (Sigma-Aldrich, catalog number: A9539 )
Bacto peptone (BD, BactoTM, catalog number: 211677 )
Bacteriological agar (VWR, catalog number: 84609.0500 )
LB broth 1.1 G per tablet (Sigma-Aldrich, catalog number: L7275-500TAB )
Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
di-Sodium hydrogen phosphate dodecahydrate (Na2HPO4•12H2O) (Merck, catalog number: 1.06579.100 )
Magnesium chloride hexahydrate (MgCl2•6H2O) (Sigma-Aldrich, catalog number: M2670-500G )
Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
Tris ultrapure (AppliChem, catalog number: A1086.1000 )
6 N HCl
Sodium acetate (NaOAc) (Merck, catalog number: 1.06268.1000 )
Silver nitrate (AgNO3) (Sigma-Aldrich, catalog number: S6506-5G )
Acetic acid (Merck, catalog number: 1.00063.2500 )
HEPES (Sigma-Aldrich, catalog number: H4034 )
Chondroitinase ABC (Amsbio, catalog number: AMS.E1028-10 ; Sigma-Aldrich, catalog number: C3667 )
Media for C. elegans growth and handling (see Recipes)
1 M CaCl2
5 N KOH
1 M MgSO4
Cholesterol solution
1 M potassium phosphate buffer pH 6.0 (KPO4)
Rich Nematode Growth medium (RNGM)
LB agar
2x YT medium
M9 buffer
Buffers for GAG purification (see Recipes)
20% ethanol
1 M MgCl2
Stock solutions
i.10% Triton X-100
ii.5 M NaCl
iii.1 M CaCl2
iv.1 M Tris-HCl
Protease buffer
DEAE elution buffer
DEAE wash buffers
DEAE wash buffer, pH 8
DEAE wash buffer, pH 4
0.1% (w/v) AgNO3
5x chondroitinase ABC buffer
2x heparinase lyase buffer
Equipment
Pipettes (for example Eppendorf, model: Research® plus )
125 ml Erlenmeyer flask (autoclaved)
Rubber bulb
10 ml glass pipettes (autoclaved, plugged)
Centrifuge
Incubator for C. elegans (Lovibond Thermostatic Cabinet)
Stereo microscope (Nikon Instruments, model: SMZ745 , eyelens: C-W10xB/22, magnification 2.5x to 50x)
Hybridization oven (Hybaid Shake ‘n’ Stack)
37 °C incubator (Heraeus Instruments)
SpeedVac (Labconco Freezer Dry System connected to Savant SpeedVac concentrator or Savant Instruments, model: SC11OA SpeedVac® Plus )
HPLC system
Merck Hitachi FL Detector L-7485 (Hitachi, model: Model L-7485 )
Interface D-7000 (Hitachi, model: Model D-7000 )
Autosampler L-7200 (Hitachi, model: Model L-7200 )
Pump L-7100 (Hitachi, model: Model L-7100 )
Column Oven L-7350 (Hitachi, model: Model L-7350 )
Reagent pumps: Shimadzu LC-10AD (Shimadzu, model: LC-10AD ); Column: Phenomenex Luna 5u C18(2) 100A (Shimadzu)
Software
D-7000 HPLC System Manager (HSM) software version 4.1
GraphPad Prism version 5.0b for Mac OS X (GraphPad Software, San Diego California USA, www.graphpad.com
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Dierker, T. and Kjellén, L. (2017). Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans. Bio-protocol 7(15): e2437. DOI: 10.21769/BioProtoc.2437.
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Category
Biochemistry > Carbohydrate > Polysaccharide
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2,438 | https://bio-protocol.org/exchange/protocoldetail?id=2438&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Digestion of Peptidoglycan and Analysis of Soluble Fragments
Ryan E. Schaub
Joseph P. Dillard
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2438 Views: 12073
Edited by: Valentine V Trotter
Reviewed by: Timo Lehti
Original Research Article:
The authors used this protocol in Dec 2016
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The authors used this protocol in:
Dec 2016
Abstract
Peptidoglycan (murein) is a vital component of the cell wall of nearly all bacteria, composed of sugars linked by short peptides. This protocol describes the purification of macromolecular peptidoglycan from cultured bacteria and the analysis of enzyme-digested peptidoglycan fragments using high performance liquid chromatography (HPLC). Digested peptidoglycan fragments can be identified by mass spectrometry, or predicted by comparing retention times with other published chromatograms. The quantitative nature of this method allows for the measurement of changes to peptidoglycan composition between different species of bacteria, growth conditions, or mutations. This method can determine the overall architecture of peptidoglycan, such as peptide stem length, the extent of cross-linking, and modifications. Muropeptide analysis has been used to study the function of peptidoglycan-associated proteins and the mechanisms by which bacteria acquire antibiotic resistance.
Keywords: Muropeptides analysis Peptidoglycan PG HPLC Muropeptide
Background
Peptidoglycan is composed of a sugar backbone linked together by peptide stems that creates a mesh-like structure important for cell shape, and turgor pressure of bacterial cells. The macromolecular peptidoglycan is assembled from monomeric units that are synthesized in the cytoplasm and consist of an N-acetylglucosamine and N-acetylmuramic acid disaccharide with a five amino acid stem. When the monomer is flipped into the periplasm, it is added to the glycan chain by transglycosylation and a portion of the peptide stems are linked together by transpeptidation.
The amino acids comprising the peptide stem can vary by species but are generally attached to muramic acid in the order L-alanine, D-glutamic acid, meso-diaminopimelic acid, D-alanine, D-alanine, with L-lysine taking the place of diaminopimelic acid in some Gram-positives. Cross-linking occurs through the free amine of the third amino acid linking either the third or fourth amino acid directly or through linker amino acids (Schleifer and Kandler, 1972). Other common modifications include amidation of amino acids (Kato and Strominger, 1968) and O-acetylation (Clarke and Dupont, 1992) or N-deacetylation (Araki et al., 1971) of sugars.
A variety of enzymes act on peptidoglycan during growth and cell division. Classes of enzymes known as lytic transglycosylases cleave glycan chains between disaccharide units at the same position as lysozyme. The important difference is that lytic transglycosylases create a 1,6-anhydro bond, in contrast to a reducing end created by lysozyme and mutanolysin. Thus the relative abundance of 1,6-anhydro bonds can be used as an approximation of glycan chain length (Ward, 1973). Different classes of peptidases act at different bonds of the peptide stem and cross-links. For example, D,D-carboxypeptidases will cleave between the fourth and fifth amino acid, while, L,D-carboxypeptidases will cleave between the third and fourth amino acid (Holtje and Tuomanen, 1991).
Muropeptide analysis can resolve the different modifications and cross-linking to give a model of the overall structure of the macromolecular peptidoglycan. One of the first observations using HPLC-based peptidoglycan analysis was the discovery that Caulobacter crescentus lacks D,D-carboxypeptidase activity (Markiewicz et al., 1983). The first comprehensive peptidoglycan analysis was done on Escherichia coli with 80 different muropeptides species identified (Glauner et al., 1988). This method has also been used to show penicillin-resistance in Neisseria meningitidis is correlated with differences in peptidoglycan structure (Antignac et al., 2003a).
Interest in peptidoglycan has seen an increase in recent years. Continued bacterial resistance to peptidoglycan-targeting antibiotics created a need for a more complete understanding of peptidoglycan metabolism. The discovery of the human peptidoglycan-recognizing proteins, NOD1 and NOD2 have also led to increased investigations into how host cells recognize peptidoglycan and how they are able to differentiate between commensal and pathogenic bacteria (Clarke and Weiser, 2011).
The following method has a number of advantages over other types of peptidoglycan analysis, including ultra-performance liquid chromatography (UPLC)-based methods. The first advantage is that nearly all of the equipment and materials are a standard part of most laboratories, so a large investment is not needed. Second, the almost 30 year history of this protocol allows comparisons with similar chromatograms to be made, allowing for preliminary identification of peptidoglycan fragments to be made quickly, then using mass spectrometry to positively identify fragments that change or are of particular interest. The third advantage is the scale of this method, which yields enough separated material for additional analysis by mass spectrometry or enzymatic reactions. More information on the history and uses of HPLC-based peptidoglycan analysis can be found in this review (Desmarais et al., 2013)
Materials and Reagents
Pipette tips
Nalgene Oak Ridge high-speed PPCO centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3119-0050 )
1.7 ml microcentrifuge tubes (MIDSCI, catalog number: AVSS1700 )
Amicon Ultra-0.5 centrifugal filter unit with ultracel-10 membrane (EMD Millipore, catalog number: UFC501024 )
Aluminum foil (Fisher Scientific, catalog number: 01-213-100 )
Filter (0.22 µm)
Bacterial growth medium (species specific)
Micrococcus lysodeikticus ATCC No. 4698, lyophilized cells (Sigma-Aldrich, catalog number: M3770 )
α-Amylase from porcine pancreas (Sigma-Aldrich, catalog number: A6255 )
Pronase protease, Streptomyces griseus (EMD Millipore, catalog number: 53702 )
1 N HCl
Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 )
Mutanolysin from Streptomyces globisporus ATCC (Sigma-Aldrich, catalog number: M9901 )
Sodium borohydride (Sigma-Aldrich, catalog number: 213462 )
Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (Fisher Scientific, catalog number: S369 )
Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: S373 )
Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-100 )
Boric acid (Acros Organics, catalog number: 327132500 )
Water (HPLC-grade) (Fisher Scientific, catalog number: W5SK-4 )
Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-1 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358 )
Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366 )
Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
o-Phosphoric acid, 85% (HPLC) (Fisher Scientific, catalog number: A260-500 )
Methanol (HPLC-grade) (Fisher Scientific, catalog number: A452SK-4 )
Trifluoroacetic acid (Sigma-Aldrich, catalog number: 302031 )
Acetonitrile (HPLC-grade) (Fisher Scientific, catalog number: A998 )
Phosphate buffer pH = 6 (PB) (see Recipes)
PB with 8% (w/v) SDS (see Recipes)
0.5 M borate buffer pH = 8 (see Recipes)
Phosphate buffered saline (PBS) (see Recipes)
HPLC separation buffer A (see Recipes)
HPLC separation buffer B (see Recipes)
HPLC desalting buffer A (see Recipes)
HPLC desalting buffer B (see Recipes)
Equipment
Kimax baffled culture flasks (Fisher Scientific, catalog numbers: 10-140-6A and 10-140-6B)
Manufacturer: DWK Life Sciences, Kimble, catalog numbers: 25630250 and 25630500 .
Pipettes (Gilson, catalog number: F167700 )
Nalgene PPCO centrifuge bottle 500 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3120-0500 )
SLA-3000 fixed angle rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 07149 )
SS-34 fixed angle rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28020 )
Sorvall RC-6 plus (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall RC 6 Plus , catalog number: 46910)
HiTemp hot water bath (Fisher Scientific, catalog number: 11-481Q )
Accumet AB150 pH benchtop meter (Fisher Scientific, model: Accumet AB150TM, catalog number: 13-636-AB150 )
UV-Vis spectrophotometer (Thermo Fischer Scientific, Thermo ScientificTM, model: GENESYSTM 10S , catalog number: 840-208100)
Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
Astec C18 HPLC column (Sigma-Aldrich, catalog number: 55024AST )
Note: This product has been discontinued.
Beckman Coulter System Gold HPLC with 126 Solvent Module, 168 Detector, and SC100 Fraction collector (Beckman Coulter, model: System Gold )
Note: This product has been discontinued.
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Schaub, R. E. and Dillard, J. P. (2017). Digestion of Peptidoglycan and Analysis of Soluble Fragments. Bio-protocol 7(15): e2438. DOI: 10.21769/BioProtoc.2438.
Download Citation in RIS Format
Category
Microbiology > Microbial biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Peptidoglycan
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2,439 | https://bio-protocol.org/exchange/protocoldetail?id=2439&type=0 | # Bio-Protocol Content
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Peer-reviewed
Wheat Coleoptile Inoculation by Fusarium graminearum for Large-scale Phenotypic Analysis
LJ Lei-Jie Jia
WW Wan-Qiu Wang
Wei-Hua Tang
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2439 Views: 10390
Reviewed by: Gazala AmeenVinay Panwar
Original Research Article:
The authors used this protocol in Dec 2012
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Dec 2012
Abstract
The ascomycete fungus Fusarium graminearum is a destructive fungal pathogen of wheat, barley and maize. Although reverse genetics and homologous recombination gene deletion methods have generated thousands of gene deletion mutants of F. graminearum, evaluating virulence of these fungal mutants is still a rate-limiting step. Here we present a protocol for inoculation of wheat coleoptiles with conidial suspensions for large-scale phenotypic analysis, and describe how it can also be used to assess fungal infectious growth and symptom developmentat a cellular scale. The inoculation method described in this protocol provides highly reproducible results in wheat coleoptile infection by F. graminearum.
Keywords: Fusarium graminearum Wheat coleoptile Plant-fungal interaction Pathogenicity assays
Background
Fusarium graminearum (previously also called Gibberella zeae) is a destructive pathogen that upon infection is responsible for causing Fusarium head blight (FHB) and seedling blight on cereal crops, as well as stalk and ear rot on maize (Dal Bello et al., 2002; Bai and Shaner, 2004; Kazan et al., 2012). Extensive molecular and genetic studies have been performed to investigate the interaction between F. graminearum and wheat. Given the availability of an efficient genetic transformation system and the well annotated genome, hundreds of F. graminearum genes have been investigated for their roles in vegetative growth, sexual development, secondary metabolism, stress responses and even virulence on host (Jia and Tang, 2015). However, only a few fungal effectors (e.g., FGL1 and deoxynivalenol) and host resistance genes (e.g., wheat Fhb1) have been identified (Proctor et al., 1995; Blümke et al., 2014; Rawat et al., 2016).
F. graminearum has been reported to lack pathogen-specialized patterns that typically induce gene-for-gene-mediated resistance in the host (van Eeuwijk et al., 1995). Furthermore, gene redundancy and functional complementation make assigning definitive virulence roles to pathogen genes achallenge. In addition, the traditional wheat head infection assay is limited due to seasonal, temporal and spatial factors. The distinct structures (rachis, paleas, lemmas, caryopses and glumes) and diverse features of wheat florets also make it difficult to track the infection progress of F. graminearum.
Previously, we used a modified wheat coleoptile infection assay and microscopic inspection to study F. graminearum infection inside host tissue (Zhang et al., 2012). Unlike the wheat head infection assay, there are few temporal and spatial constraints for the seedling infection system. The wheat coleoptile infection assay is performed in a growth chamber that can hold up to two hundred 24-well plates, which means more than 100 genes can be evaluated for their roles in virulence (three independent transgenic lines for each tested gene, and at least twelve seedlings for inoculation of each fungal strain). The time required to complete the assay is short: ten days for seed germination (Figure 1), inoculation (Figure 2) and examination of lesion size (Figure 3). The structure of wheat coleoptile is simple: the annular coleoptile comprises similar cells and two vascular bundles (Figure 2D) and is easy to inspect microscopically (Zhang et al., 2012). Seven genes were identified required for full virulence of F. graminearum on wheat coleoptile, and several of which were also required for wheat head infection (Zhang et al., 2012) and even maize stalk infection (Zhang et al., 2016).
Materials and Reagents
Pipette tips (Corning, Axygen®, catalog number: T-300-R-S )
Disposable paper towels (Vinda Classic Blue 1800, Vinda, catalog number: V4028 )
24-well cell culture plate (Corning, Costar®, catalog number: 3524 )
Sterile toothpick
Microtubes (Corning, Axygen®, catalog number: MCT-150-C )
Medical-grade gauze (regular cotton yarn, Shanghai Honglong Medical Material Company)
Medical-grade absorbent cotton (Shanghai Honglong Medical Material Company)
Enamel tray (20x 30 x 5 cm)
Glass slides (specifications: 76.2 x 25.4 mm, thickness: 1.0-1.2 mm) (Livingstone, catalog number: 7105-1 )
Cover slips (specifications: 24 x 50 mm, thickness: 0.13-0.16 mm) (CITOTEST LABWARE MANUFACTURING, catalog number: 10212450C )
Fungal strains: F. graminearum wild-type strain PH-1 (NRRL 31084), AmCyanPH-1 (Zhang et al., 2012) and gene deletion mutants of PH-1 (Zhang et al., 2012 and 2016)
Plant material: Wheat (Triticum aestivum) cultivar Zhongyuan 98-68 (susceptible to F. graminearum and widely cultured in Henan, China)
Sterile water
V8 vegetable juice (CAMPBELL, catalog number: V8® ORIGINAL )
Calcium carbonate (CaCO3)
Agar powder
Mung beans
V8 juice agar medium (He et al., 2016; see Recipes)
Mung bean liquid medium (He et al., 2016; see Recipes)
Equipment
500 ml flask
Pipettes (Eppendorf)
Growth chamber (Ningbo Jiangnan Instrument Factory, model: RXZ-1000 )
Mould cultivation cabinet (Yiheng, model: MJ-150-I )
Biological safety cabinet (ESCO Micro, model: FHC-1200A )
Constant temperature shaker (Taicang, model: DHZ-DA )
Hemocytometer (0.10 mm, 1/400 mm2) (QIUJING, model: XB-K-25 )
Fluorescent microscope (Olympus, model: Olympus BX51 )
Confocal microscope (Olympus, model: Fv10i )
Centrifuge (Beckman Coulter, model: Avanti J-E Series )
Camera (Canon, model: EOS 7D )
Autoclave
Rule
Software
ImageJ (http://rsbweb.nih.gov/ij/index.html)
Microsoft Excel
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Jia, L., Wang, W. and Tang, W. (2017). Wheat Coleoptile Inoculation by Fusarium graminearum for Large-scale Phenotypic Analysis. Bio-protocol 7(15): e2439. DOI: 10.21769/BioProtoc.2439.
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Category
Plant Science > Plant immunity > Disease bioassay
Microbiology > Microbe-host interactions > Fungus
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244 | https://bio-protocol.org/exchange/protocoldetail?id=244&type=0 | # Bio-Protocol Content
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A Quick, No Frills Approach to Mouse Genotyping
Manuel E. Lopez
Published: Vol 2, Iss 15, Aug 5, 2012
DOI: 10.21769/BioProtoc.244 Views: 26428
Original Research Article:
The authors used this protocol in Mar 2011
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Abstract
Mice are extremely powerful mammalian genetic model organisms for basic and medical research, but managing a colony of transgenic mice is time consuming and expensive, many times requiring the help of dedicated technicians. Slow and laborious genotyping procedures add to the hassle. Outsourcing is costly and may not be as fast as desired, especially when setting up time sensitive experiments. Ultrafast genotyping protocols often require real-time PCR instruments and commercial reagents that may not be economical or practical. This protocol, adapted from methods suggested by The Jackson Laboratory, employs a minimalist approach that maximizes convenience by simplifying the tissue digestion/DNA extraction process and using a high-speed electrophoresis system for sample analysis. Genotype PCR results can be obtained in 3 h or less for as many samples as can fit in a PCR machine or can be efficiently handled by a user. Subsequent ethanol or chloroform purified DNA can be used in a standard PCR reaction to roughly identify a homozygous and a hemizygous mouse.
Materials and Reagents
NaOH (NaOH pellets)
Taq DNA Polymerase with ThermoPol buffer (New England Biolabs, catalog number: M0267X *, M0267L , or M0267S )
* Note: At 4,000U, ~800 μl of Taq serves several thousand PCR reactions. Buffer becomes a limiting reagent. ThermoPol Buffer recipe is available at NEB website. This buffer can be ordered separately from NEB (New England Biolabs, catalog number: B9004S )
Primers, recommend to be 18-21 bp in length, have a melting temperature above 56 °C and around 58 °C, and produce amplicons of 150-600 bp.
DNA loading buffer
Recommend Orange G (Sigma-Aldrich, catalog number: O3756 ) instead of bromophenol blue for loading dye
DNA ladder range for 100-800 bp range
Recommend 1 kb Plus DNA ladder (Life Technologies, catolog number: 10787-018 )
Agarose
EtBr (Sigma-Aldrich, catalog number: E8751 ) or SYBR Safe DNA Gel Stain (Life Technologies, catalog number: S33102 )
NaOAc
EtOH
Phenol/Chloroform/Isoamyl alcohol (25: 24: 1) (Do not use acid phenol)
50 mM NaOH in dH2O (see Recipes)
10 mM dNTP Mix (see Recipes)
1x TAE buffer (see Recipes)
dNTP set 100 mM each A, C, G, T (GE Life Sciences, catalog number: 28406552 ) (see Recipes)
0.3 M NaOAc in ddH2O (see Recipes)
Equipment
PCR Thermal cycler (96 well capacity preferred)
Centrifuges (mini for PCR tubes and microcentrifuge for 1.5 ml tubes)
Liberty 1 buffer-less high speed gel system (Neuvitro, 6Mgel - SYS-LBT1)
Liberty 1, 12-channel pipette compatible 13 teeth combs (Neuvitro, 6Mgel - CMS-1315)
Multichannel Pipette 2-20 μl
Repeat Pipettor 10-125 μl
PCR tube with cap, 8 or 12 PCR strip tubes, or 96-well PCR plate
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Molecular Biology > DNA > Electrophoresis
Molecular Biology > DNA > Genotyping
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2,440 | https://bio-protocol.org/exchange/protocoldetail?id=2440&type=0 | # Bio-Protocol Content
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A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae
VB Vidhya Bharathi
AG Amandeep Girdhar
Basant K Patel
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2440 Views: 10714
Edited by: Yanjie Li
Reviewed by: Sandeep Dave
Original Research Article:
The authors used this protocol in Dec 2016
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Dec 2016
Abstract
The yeast Saccharomyces cerevisiae (S. cerevisiae) harboring ade1 or ade2 mutations manifest red colony color phenotype on rich yeast medium YPD. In these mutants, intermediate metabolites of adenine biosynthesis pathway are accumulated. Accumulated intermediates, in the presence of reduced glutathione, are transported to the vacuoles, whereupon the development of the red color phenotype occurs. Here, we describe a method to score for presence of oxidative stress upon expression of amyloid-like proteins that would convert the red phenotype of ade1/ade2 mutant yeast to white. This assay could be a useful tool for screening for drugs with anti-amyloid aggregation or anti-oxidative stress potency.
Keywords: Amyloid Oxidative stress ROS Yeast ade1 mutant TDP-43 DCFDA
Background
The yeast Saccharomyces cerevisiae cells mutant for ADE1 or ADE2 genes (e.g., ade1∆, ade2∆, ade1-14, ade2-1), when grown on YPD (Yeast Peptone Dextrose) medium, accumulate red pigment in the vacuole which is an intermediate metabolite of adenine biosynthesis pathway (Sharma et al., 2003). The suppressible allele ade1-14 which contains a premature stop codon, has been widely used to score for [PSI+] prion state of the translation termination factor Sup35 protein. In a [psi-] yeast, the Sup35p remains soluble and functional, therefore translation is terminated efficiently at the premature stop codon of the ade1-14 allele leading to synthesis of truncated and non-functional Ade1 protein. Thus, the adenine biosynthesis cascade remains incomplete leading to accumulation of intermediate metabolite yielding the red phenotype of the [psi-] yeast. In contrast, in a [PSI+] yeast, the Sup35p is aggregated and partially inactivated, thereby causing read-through of the premature non-sense codon of the ade1-14 allele that leads to functional Ade1 protein synthesis. Thus, adenine biosynthesis pathway is completed and no red intermediate metabolite is accumulated, consequently giving the [PSI+] cells, a white phenotype (Chernoff et al., 1993). In the view that the red color development from this adenine biosynthesis intermediate metabolite requires presence of reduced glutathione(Sharma et al., 2003), we reasoned that presence of oxidative stress which would oxidize glutathione, would also cause white color conversion alike to the white phenotype of the [PSI+] yeast. Additionally, it is known that the aggregations of several amyloid proteins cause cellular oxidative stress, therefore we attempted to develop a red/white reporter color assay for amyloid-induced oxidative stress in the ade1/ade2 mutant yeast background similar to the widely used red/white assay for the [psi-] to [PSI+] conversion (Bharathi et al., 2016). We present here red/white color switch assay using two amyloid-like proteins, TAR DNA binding protein 43 (TDP-43) and Fused in Sarcoma (FUS), both of which are implicated in the pathogenesis of the motor neuron disease, Amyotrophic Lateral Sclerosis (ALS) (Rossi et al., 2016). Such simplistic color assay for amyloid-induced oxidative stress in yeast has never been reported previous to our Bharathi et al., 2016 manuscript and has the potential to be a highly useful methodology to study for amyloid protein-induced toxicity in yeast.
Materials and Reagents
Pipette tips (Tarsons products pvt ltd., India)
Microcentrifuge tubes (Tarsons products pvt ltd., India)
Petri dishes (Genaxy scientific pvt ltd., India)
Sterile toothpick
Yeast Saccharomyces cerevisiae strain: 74D-694 (MATa ade1-14, his3-200, ura3-52, leu2-3, 112, trp1-289, [psi-])
Note: This assay can also be performed using an ade2-1 allele bearing mutant yeast or an ade2∆ mutant yeast as well as using an ade1∆ mutant yeast.
Plasmids
pRS416-GAL1p-FUS-YFP (URA3) (Addgene, catalog number: 29593 )
pRS416-GAL1p-TDP43-YFP (URA3) (Addgene, catalog number: 27447 )
pRS416 (URA3)
pAG416 GAL1p-ccdB-EGFP (URA3) (Addgene, catalog number: 14195 )
0.1 M phosphate buffer (pH 7.4)
2’,7’-Dichlorofluoroscein diacetate [DCFDA] (Sigma-Aldrich, catalog number: D6883 )
Lithium acetate (Sigma-Aldrich, catalog number: 517992 )
Peptone (HiMedia Laboratories, catalog number: RM001 )
D-glucose [Dextrose] (AMRESCO, catalog number: 0188 )
Yeast extract (HiMedia Laboratories, catalog number: RM027 )
Bacteriological agar (HiMedia Laboratories, catalog number: RM026 )
D-raffinose (Sigma-Aldrich, catalog number: 83400 )
D-galactose (Sigma-Aldrich, catalog number: 48260 )
Yeast nitrogen base (HiMedia Laboratories, catalog number: G090 )
Sodium dodecyl sulphate [SDS] (Sigma-Aldrich, catalog number: L3771 )
Chloroform (Sigma-Aldrich, catalog number: 372978 )
Ammonium sulfate (Sigma-Aldrich, catalog number: A2939 )
Amino acids: Arginine, Histidine, Isoleucine, Valine, Lysine, Methionine, Adenine, Phenylalanine, and Tyrosine (HiMedia Laboratories, India); Leucine and Tryptophan (Sigma-Aldrich, USA)
YPD medium (see Recipes)
SRaf-Ura + 0.1% gal + ¼ YP plate (see Recipes)
SRaf-Ura broth (see Recipes)
SD-Ura + ¼ YP plate (see Recipes)
SD-Ura + ⅓ YP plate (see Recipes)
DCF extraction buffer (see Recipes)
Equipment
Pipettes (Corning, USA)
250 ml conical flasks (Borosil Glass Works ltd., India)
Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: MicroCL 21R )
Fluorescence microscope (Leica Microsystems, model: Leica DM2500 )
Laminar flow biosafety cabinet (Esco Micro, model: ACB-4A1 )
Temperature controlled incubator (JS Research, model: JSGI-100T )
Temperature controlled orbital shaker (Eppendorf, New BrunswickTM, model: Excella® E24 )
UV-Vis spectrophotometer (Hitachi High-Technologies, model: U-3900 )
Vortex mixer (Remi, model: CM-101 )
Autoclave sterilizer (JS Research, model: JSAC-100 )
Multimode microplate reader (Molecular Devices, model: Spectramax M5e )
Camera
Software
GraphPad QuickCalcs software (from GraphPad Software Inc., USA)
https://www.graphpad.com/quickcalcs/ttest1.cfm
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bharathi, V., Girdhar, A. and Patel, B. K. (2017). A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae. Bio-protocol 7(15): e2440. DOI: 10.21769/BioProtoc.2440.
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Category
Microbiology > Microbial metabolism > Other compound
Microbiology > in vivo model > Fungi
Cell Biology > Cell-based analysis > Extracellular microenvironment
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2,441 | https://bio-protocol.org/exchange/protocoldetail?id=2441&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis
KH Kyohei Higashi
TT Toshihiko Toida
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2441 Views: 6523
Reviewed by: Hui Zhu
Original Research Article:
The authors used this protocol in Sep 2016
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Sep 2016
Abstract
Glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), heparin (HP), heparan sulfate (HS) and keratan sulfate (KS) are linear, sulfated repeating disaccharide sequences containing hexosamine and uronic acid (or galactose in the case of KS). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S(β1-3)Galactose(β1-]-branched CS-E was identified from the clam species M. chinensis. Here, we report the isolation protocol for KS-branched CS from M. chinensis.
Keywords: Mactra chinensis Glycosaminoglycan Chondroitin sulfate Keratan sulfate Galactose
Background
GAGs are found in tissues as the glycan moieties of proteoglycan (PG) glycoconjugates. CS is a GAG type composed of linear, sulfated repeating disaccharide sequences of N-acetyl-D-galactosamine (GalNAc) and glucuronic acid (GlcA). Another GAG type, DS, is biosynthesized through the action of glucuronyl C5-epimerase on CS, converting its GlcA to the CS epimer, iduronic acid (IdoA). The other GAG types HS and HEP are consisted of sulfated repeating disaccharide sequence of N-acetyl-D-glucosamine (GlcNAc) and GlcA/IdoA. KS is composed of sulfated repeating disaccharide sequences of GlcNAc and galactose. Among them, structural variations of CS, such as sulfation patterns and fucosylation, depend on the species and tissue of origin. For example, the A-unit with the structure [-4)GlcA(β1-3)GalNAc4S(β1-] (where S designates a sulfonate residue) is a predominant disaccharide found in mammalian or chicken tracheal cartilage CS, while the C-unit with the structure [-4)GlcA(β1-3)GalNAc6S(β1-] is a major disaccharide found in shark cartilage or salmon nasal cartilage CS. In contrast, a significant amount of the D-unit disaccharide [-4)GlcA2S(β1-3)GalNAc6S(β1-] is characteristically found in CS isolated from shark cartilage, while the E-unit disaccharide [-4)GlcA(β1-3)GalNAc4S,6S(β1-] is characteristic in squid cartilage. In sea cucumber, a sulfated fucose branches at the 3-OH position of GlcA. The disaccharide composition of CS governs its biological activities, including cell proliferation, migration, differentiation, cell-cell crosstalk, adhesion and wound repair through the interaction with growth factors, receptors, and other CS-binding proteins. Evidence suggests that CS structure is tightly correlated with function. For example, consecutive and disulfated disaccharide units including B, D and E-units in CS are critical for the interaction between CS and binding proteins (Hikino et al., 2003). A branched fucose at the 3-OH position of GlcA is also required for the anticoagulant activity of fucosylated CS (Mourão et al., 1996).
In general, isolation of GAGs is carried out as follows. 1) acetone defatting, 2) proteolysis, 3) collection of the GAGs, 4) fractionation of GAGs by anion-exchange chromatography and 5) desalting (Maccari et al., 2015). In our protocol, actinase E from Streptomyces griseus (step 2) and cetylpyridinium chloride precipitation (step 3) were used for the isolation of GAGs.
Materials and Reagents
200 and 1,000 μl pipette tips (Thermo Fisher Scientific)
Centrifuge tube
Spectra/Por®7 Dialysis Membrane Pre-treated RC Tubing MWCO: 3,500 (Spectrum, catalog number: 132111 )
HiPrepTM DEAE FF 16/10 (GE Healthcare, catalog number: 28936541 )
Dry powder of hot water extract from M. chinensis viscera obtained from Futtsu City Fishery Association in Chiba, Japan
Acetone (Wako Pure Chemical Industries, catalog number: 011-00357 )
Acetic acid (NACALAI TESQUE, catalog number: 00212-43 )
Perchloric acid (60%, w/v) (NACALAI TESQUE, catalog number: 26502-85 )
Ethanol (99.5% w/v) (NACALAI TESQUE, catalog number: 14713-53 )
Tris (hydroxymethyl)aminomethane (Tris) (NACALAI TESQUE, catalog number: 35406-91 )
Actinase E (Funakoshi, catalog number: KA-001 )
Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
Sodium tetrahydroborate (Wako Pure Chemical Industries, catalog number: 192-01472 )
Cetylpyridinium chloride monohydrate (99.0-102.0% w/v) (Wako Pure Chemical Industries, catalog number: 086-06683 )
Hydrochloric acid (HCl) (35.0% w/v) (NACALAI TESQUE, catalog number: 18321-05 )
Sodium chloride (NaCl) (NACALAI TESQUE, catalog number: 31320-05 )
Sodium dihydrogenphosphate, anhydrous (NaH2PO4) (NACALAI TESQUE, catalog number: 31720-65 )
Di-sodium hydrogenphosphate (Na2HPO4) (NACALAI TESQUE, catalog number: 31801-05 )
Tris acetate buffer (pH 8.0) (see Recipes)
NaOH buffer (see Recipes)
Cetylpyridinium chloride solution (see Recipes)
50 mM sodium phosphate (pH 6) (see Recipes)
2.0 M NaCl in 50 mM sodium phosphate (pH 6) (see Recipes)
Equipment
Refrigerated centrifuge (Sakuma, model: M200-IVD )
Fume hood
Erlenmeyer flask (AGC, catalog number: 4980FK500 )
Stainless steel spoon (180 mm)
Pipettes (Gilson, models: P20 , P200 and P1000 )
Corning® reusable low form beaker, polypropylene, size 3 L (Corning, catalog number: 1000P-3L )
Stir bar
BioShaker (TAITEC, model: BR-23FP MR )
Freeze drier (TOKYO RIKAKIKAI, Eyela, model: FDU-830 )
Gradient pump (Bio-Rad Laboratories, model: ECONO GRADIENT PUMP )
Analytical balance (Shimadzu, model: ATX224 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Higashi, K. and Toida, T. (2017). Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis. Bio-protocol 7(15): e2441. DOI: 10.21769/BioProtoc.2441.
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Category
Biochemistry > Carbohydrate > Polysaccharide
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2,442 | https://bio-protocol.org/exchange/protocoldetail?id=2442&type=0 | # Bio-Protocol Content
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Peer-reviewed
CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum
Marianne Nymark
AS Amit Kumar Sharma
MH Marthe C. G. Hafskjold
TS Torfinn Sparstad
AB Atle M. Bones
PW Per Winge
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2442 Views: 13167
Edited by: Dennis Nürnberg
Reviewed by: Vera Karolina SchoftAgnieszka Zienkiewicz
Original Research Article:
The authors used this protocol in May 2016
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May 2016
Abstract
The establishment of the CRISPR/Cas9 technology in diatoms (Hopes et al., 2016; Nymark et al., 2016) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of areas ranging from oceanography to materials science, in nano- and environmental biotechnology, and are presently being investigated as a source of renewable carbon-neutral fuel and chemicals. Here we present a detailed protocol of how to perform CRISPR/Cas9 gene editing of the marine diatom Phaeodactylum tricornutum, including: 1) insertion of guide RNA target site in the diatom optimized CRISPR/Cas9 vector (pKS diaCas9-sgRNA), 2) biolistic transformation for introduction of the pKS diaCas9-sgRNA plasmid to P. tricornutum cells and 3) a high resolution melting based PCR assay to screen for CRISPR/Cas9 induced mutations.
Keywords: CRISPR/Cas9 technology Diatoms Phaeodactylum tricornutum Biolistic transformation HRM analyses
Background
The CRISPR/Cas9 system has proven to be a very efficient and successful genome editing system in a number of eukaryotic organisms, now also including microalgae (Hopes et al., 2016; Nymark et al., 2016; Shin et al., 2016). The CRISPR/Cas9 system includes a guide RNA (gRNA) and a nuclease called Cas9 (Sander and Joung, 2014). These two molecules form a complex where the gRNA directs the complex to the target of interest. The Cas9 nuclease induces double strand brakes at the target site that can be repaired by nonhomologous end joining (NHEJ) which can result in indel mutations, or via the homology-directed repair (HDR) pathway that can be exploited to create defined alterations of the DNA. The presented protocol is the first to describe a step-by-step procedure for applying the CRISPR/Cas9 system to create gene-targeted mutations (indels ranging from one to hundreds of nucleotides) in one of the main diatom model species, P. tricornutum.
Materials and Reagents
Pipette tips, 1,000 µl (SARSTEDT, catalog number: 70.762.100 )
Pipette tips, 200 µl (SARSTEDT, catalog number: 70.760.502 )
Pipette tips, 20 µl (Biosphere® Tip 20 µl neutral) (SARSTEDT, catalog number: 70.1116.200 )
Filter tips, 20 µl (Biosphere® Fil. Tip 20 µl neutral) (SARSTEDT, catalog number: 70.1116.210 )
24 or 48-cell multiwell cell culture plates, flat bottom, TC treated (VWR, catalog number: 734-2325 or 734-2326 )
1.5 ml tube
Parafilm
50 ml centrifuge tube (SARSTEDT, catalog number: 62.547.254 )
0.2 µm sterile filter
Macrocarriers (Bio-Rad Laboratories, catalog number: 1652335 )
1,550 psi rupture discs (Bio-Rad Laboratories, catalog number: 1652331 )
Stopping screens (Bio-Rad Laboratories, catalog number: 1652336 )
Phaeodactylum tricornutum cells (NCMA Bigelow Laboratory for Ocean Sciences, Bigelow, catalog number: CCMP2561 starter culture)
Competent DH5α E.coli cells (‘home-made’ RbCl competent cells [efficiency ≥ 1.0 x 106 cfu/µg])
pKS diaCas9-sgRNA plasmid (Addgene, catalog number: 74923 )
pAF6 plasmid (Falciatore et al., 1999) containing the ShBle gene conferring resistance to zeocin
BsaI-HF restriction endonuclease (New England Biolabs, catalog number: R3535S )
Complementary oligos (24 nt) with 5’ TCGA and AAAC overhangs (for creation of the adapter for targeting the gene of interest). Custom DNA oligos can be ordered from Sigma-Aldrich
Wizard® SV Gel and PCR Clean-Up Kit (Promega, catalog number: A9282 )
T4 DNA ligase buffer (New England Biolabs, catalog number: M0202S )
ExTaq DNA polymerase and buffer system (AH Diagnostics) (Takara Bio, catalog number: RR001A )
QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106 )
50% (v/v) seawater plates supplemented with f/2-Si, 1% (w/v) agar plates
Tungsten M10 or M17 microcarriers (Bio-Rad Laboratories, catalog number: 1652266 or 1652267 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
Spermidine (Sigma-Aldrich, catalog number: S2626 )
70% (v/v) and 100% EtOH
70% (v/v) isopropanol
50% (v/v) seawater plates supplemented with f/2-Si, 1% (w/v) agar plates, 100 µg/ml zeocin
Zeocin (InvivoGen, catalog number: ant-zn-5p )
TOPO® TA Cloning® Kit for Sequencing (Thermo Fisher Scientific, InvitrogenTM, catalog number: 450030 )
LightCycler 480 High Resolution Melting Master Kit (Roche Molecular Systems, catalog number: 04909631001 )
TritonTM X-100 (Sigma-Aldrich, catalog number: X100 )
Trizma® base (Sigma-Aldrich, catalog number: T6066 )
Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E5134 )
Pancreatic peptone (VWR, catalog number: 26208.297 )
Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
Bacteriological agar (VWR, catalog number: 84609.5000 )
Sodium chloride (NaCl) (Merck, catalog number: 106404 )
Lysis buffer (Buffer for lysis of P. tricornutum cells) (see Recipes)
LB medium (see Recipes)
LB agar plates (see Recipes) containing 100 µg/ml ampicillin
f/2 growth medium with natural (or artificial) seawater (see Recipes) (Guillard, 1975)
Equipment
NanoDrop ND-1000 or ND-2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 1000 or NanoDropTM 2000 )
Heating block (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88870005 )
Water bath (Grant Instruments)
Micropipettes (Eppendorf, model: Eppendorf Research® , variable volume)
Table top centrifuges (Fisher Scientific, model: accuspinTM Micro 17R , catalog number: 13-100-676)
Incubator shaker (Eppendorf, New BrunswickTM, model: Innova® 44 , catalog number: M1282-0002)
Sterile bench (Thermo Fisher Scientific, Thermo ScientificTM, model: Holten Horizontal Laminar Airflow )
PCR thermal cycler (Bio-Rad Laboratories, model: T100TM, catalog number: 1861096 )
LightCycler® 96 Real-Time PCR system (Roche Molecular Systems, catalog number: 05815916001 )
Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, catalog numbers: 165-2257 and 165-2250LEASE to 165-2255LEASE )
Vortex
Autoclave
Refrigerator
Software
LightCycler® 96 Software Version 1.1 (Roche Molecular Systems)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Nymark, M., Sharma, A. K., Hafskjold, M. C. G., Sparstad, T., Bones, A. M. and Winge, P. (2017). CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum. Bio-protocol 7(15): e2442. DOI: 10.21769/BioProtoc.2442.
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Category
Microbiology > Microbial genetics > Mutagenesis
Molecular Biology > DNA > Mutagenesis
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2,443 | https://bio-protocol.org/exchange/protocoldetail?id=2443&type=0 | # Bio-Protocol Content
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Peer-reviewed
Assessment of Modulation of Protein Stability Using Pulse-chase Method
ME Mohamed Elgendy
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2443 Views: 10770
Edited by: Jia Li
Reviewed by: Mohan TC
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
Pulse-chase technique is a method widely used to assess protein or mRNA stability. The principle of pulse-chase relies on labeling proteins or mRNA produced during a short period of time called ‘pulse’ and then following the rate of disappearance of those labeled proteins over a period of time called ‘chase’. This technique thus allows quantitative analysis of modulation of protein or mRNA stability under different treatments or culturing conditions.
Keywords: Pulse-chase Protein stability Half-life Protein degradation Proteasome Lysosome MCL-1 mTOR Sunitinib
Background
Pulse-chase technique is a method that involves culturing cells in a medium containing labeled amino acids for a short period of time known as ‘pulse’. This results in the generation of newly-synthesized polypeptides incorporating labeled amino acids. The pulse step is followed by a ‘chase’ step in which labeling medium is washed out to terminate the labeling process and is replaced by a medium with non-labeled amino acids to allow for the quantification of the initially synthesized radiolabeled proteins at any given time during this chase phase. This technique thus allows quantitative analysis of the processing of a protein of interest from synthesis to degradation in a timely fashion. Pulse-chase method can be used to analyze a variety of processes such as protein folding, co-translational modifications and intracellular transport (Jansens and Braakman 2003; Magadán, 2014). However, pulse-chase technique is most commonly used to assess the stability of a protein under different experimental conditions.
Radioactivity is a commonly used label. Labeling is usually done for proteins using radioactive S35 Methionine. The kinetics of disappearance of the radiolabeled proteins relies on how fast these proteins get degraded which can be exploited to examine the effect of different experimental conditions on protein stability. To assess the stability of a particular protein of interest, this protein is immunoprecipitated from all other immunolabeled proteins that were produced during the pulse period using a specific antibody. Immunoprecipitation will isolate a ratio of radiolabeled protein that was produced during the pulse and has not been degraded until the time of immunoprecipitation. Then radioactivity is measured to assess the relative amounts of labeled protein over a period of time.
While the focus of this protocol is the assessment of protein stability, pulse-chase method can also be used to assess the stability of mRNA. Labeling of mRNA during the pulse step can be done using 3UTP. Quantitative analysis of mRNA of interest then follows by taking aliquots of total mRNA at different time points and applying one of two methods: 1) Dot blot method in which the labeled mRNA of interest anneals to a complementary single stranded nucleic acid attached to a filter paper, other labeled mRNA are then washed out and radioactivity is measured by autoradiogram or scintillation counting, 2) Affinity purification method in which the labeled mRNA of interest anneals to a complementary anchored RNA/DNA immobilized to beads, the beads are then collected by centrifugation and radioactivity of the captured labeled mRNA is quantified.
Materials and Reagents
Other than the materials routinely used for mainlining cells in tissue culture and for immunoprecipitation and SDS-PAGE, the specific reagents required for the pulse-chase experiment are:
Pipette tips
1.5 ml micro-centrifuge tubes
Cell culture dishes
Plastic cell scraper
Tissue culture medium, appropriate for the cell line used, without methionine and cysteine
DPBS
Dithiothreitol (DTT)
Protease inhibitor cocktail
Phosphatase inhibitor cocktail (Sigma-Aldrich, catalog number: P2850 )
N-Ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: R3876 )
Note: This product has been discontinued.
Bradford colorimetric assay
Protein A-Sepharose bead
[35S]-Methionine (PerkinElmer, catalog number: NEG709A500UC )
Methionine (250 mM in H2O, store at -20 °C) (Sigma-Aldrich, catalog number: M5308 )
Cysteine (500 mM in H2O, store at -20 °C) (Sigma-Aldrich, catalog number: C7352 )
Sodium chloride (NaCl)
Triton X-100
Ethylenediaminetetraacetic acid (EDTA)
Glycerol
Tris-HCl
Sodium dodecyl sulfate (SDS)
Methanol
Acetic acid
Pulse (labeling) medium (see Recipes)
Chase medium (see Recipes)
Lysis buffer (see Recipes)
Wash buffer (see Recipes)
Fixation solution (see Recipes)
Equipment
Pipettes
Refrigerated centrifuge
95 °C heat block
Cell culture incubator (37 °C humidified 5% CO2)
SDS-PAGE apparatus
Radiographic cassette
-80 °C freezer
Ice buckets
37 °C water bath
Aspiration flasks (Safety-approved for radiolabeled materials)
Head over head rotator
A gel drying equipment
Software
ImageJ
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Elgendy, M. (2017). Assessment of Modulation of Protein Stability Using Pulse-chase Method. Bio-protocol 7(16): e2443. DOI: 10.21769/BioProtoc.2443.
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Category
Molecular Biology > Protein > Stability
Cell Biology > Cell-based analysis > Protein synthesis
Biochemistry > Protein > Immunodetection
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2,444 | https://bio-protocol.org/exchange/protocoldetail?id=2444&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation and Analysis of Stromal Vascular Cells from Visceral Adipose Tissue
JV Jessica Vu
WY Wei Ying
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2444 Views: 12759
Edited by: Jia Li
Original Research Article:
The authors used this protocol in Mar 2017
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Abstract
The obesity epidemic is the underlying driver of the type 2 diabetes mellitus epidemic. A remarkable accumulation of various pro-inflammatory immune cells in adipose tissues is a hallmark of obesity and leads to pathogenesis of tissue inflammation and insulin resistance. Here, we describe a detailed protocol to isolate adipose tissue stromal vascular cells (SVCs), which enrich various immune cells of adipose tissues. These SVCs can be used to examine the population and activation status of immune cells by tracking their cell surface antigens, gene expression, and activation of specific signaling pathways.
Keywords: Adipose tissue Stromal vascular cell Collagenase digestion Immune cell Flow cytometry analysis
Background
Over the past several decades, obesity is now an epidemic and has become one of the most common causes of insulin resistance. Insulin resistance is the key etiology for the pathogenesis of metabolic syndrome. Prolonged status of metabolic syndrome drives the development of type 2 diabetes mellitus (T2DM) (Romeo et al., 2012; Johnson and Olefsky, 2013; Saltiel and Olefsky, 2017).
Chronic low-degree tissue inflammation, accompanied by enhanced immune cell infiltration, is a hallmark of obesity in both rodent and human and is a major causal factor for the pathogenesis of insulin resistance through promoting the inflammation status and interrupting the insulin signalling (Romeo et al., 2012; Johnson and Olefsky, 2013; Saltiel and Olefsky, 2017). The infiltrated immune cells such as pro-inflammatory macrophages and B cells play critical roles in modulating obesity-associated adipose tissue inflammation and insulin resistance (Weisberg et al., 2003; Winer et al., 2011). Chronic nutrient excess drives adipose tissue macrophages (ATMs) to undergo a unique phenotypic switch from anti-inflammatory M2-like activation in lean adipose tissue to a more pro-inflammatory M1-like activation state in obese tissues (Lumeng et al., 2007; Nguyen et al., 2007; Lumeng et al., 2008). Pro-inflammatory M1-like ATMs contribute to the development of tissue inflammation and systemic insulin resistance in obesity. Our recent study also demonstrates that leukotriene B4 (LTB4)-induced recruitment and activation of adipose tissue B2 (ATB2) cells can cause obesity-induced insulin resistance (Ying et al., 2017). In this protocol, we provide a step-by-step procedure to isolate stromal vascular cells from adipose tissue and characterize various immune cells in adipose tissues.
Materials and Reagents
Pipette tips (USA Scientific)
100-mm Petri dish
50 ml Falcon tube (Corning, Falcon®, catalog number: 352070 )
Nylon biopsy bag (Electron Microscopy Sciences, catalog number: 62324-35 )
MicroAmp Optical 96-well reaction plate (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8010560 )
Stromal vascular cells (SVCs)
70% ethanol
BDTM stabilizing fixative buffer (BD, BD Biosciences, catalog number: 339860 )
Phosphate-buffered saline (PBS)
2% fetal bovine serum (FBS)
Antibody
Rabbit monoclonal anti-GAPDH (Cell Signaling Technology, catalog number: 5174 )
Rabbit monoclonal anti-Phospho-NF-κB p65 (Cell Signaling Technology, catalog number: 3033 )
PE-Cyanine7 anti-mouse F4/80 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 25-4801-82 )
Alexa Fluor 488 anti-mouse CD11b (Thermo Fisher Scientific, eBioscience TM, catalog number: 53-0112-82 )
APC anti-mouse CD11c (Thermo Fisher Scientific, eBioscienceTM , catalog number: 17-0114-82 )
PE anti-mouse CD206 (BioLegend, catalog number: 141706 )
eVolve-605 anti-mouse CD45 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 83-0451-42 )
APC anti-mouse CD19 (Thermo Fisher Scientific, eBioscienceTM , catalog number: 17-0193-82 )
Trizol reagent (Thermo Fisher Scientific, InvitrogenTM , catalog number: 15596026 )
Direct-zol RNA kits (Zymo Research, catalog number: R2070 )
High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4368813 )
qPCR primers (Table 1)
Table 1. qPCR primer information
SYBR Green PCR Master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4309155 )
Hanks’ balanced salt solution (HEPES) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
Collagenase II (Sigma-Aldrich, catalog number: C1764-50MG )
Bovine serum albumin (BSA)
Ammonium chloride (NH4Cl)
Potassium bicarbonate (KHCO3)
5% EDTA
Sodium azide (NaN3)
Iscove’s Modified Dulbecco’s Medium (IMDM)
Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number. 15140122 )
Digestion buffer (see Recipes)
Red blood cell lysis buffer (see Recipes)
FACS staining buffer (see Recipes)
Complete culture medium (see Recipes)
Equipment
Pipettes
Mortar and pestle
Curved scissors
New Brunswick Scientific 12400 incubator shaker (Eppendorf, model: New BrunswickTM 124 )
Eppendorf centrifuge 5810R (Eppendorf, model: 5810 R )
TC20 automated cell counter (Bio-Rad Laboratories, model: TC20TM, catalog number: 1450102 )
BD FACSCanto flow cytometry analyzer
StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOnePlusTM , catalog number: 4376600)
DNA Engine Peltier Thermal Cycler (Bio-Rad Laboratories, model: PTC-200 )
NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 1000 )
Software
FlowJo
GraphPad Prism
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Vu, J. and Ying, W. (2017). Isolation and Analysis of Stromal Vascular Cells from Visceral Adipose Tissue. Bio-protocol 7(16): e2444. DOI: 10.21769/BioProtoc.2444.
Download Citation in RIS Format
Category
Immunology > Immune cell isolation > Stromal vascular cell
Cell Biology > Cell isolation and culture > Cell isolation
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2,445 | https://bio-protocol.org/exchange/protocoldetail?id=2445&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes
YA Yannick O. Alexandre
Scott N Mueller
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2445 Views: 9942
Edited by: Ivan Zanoni
Reviewed by: Meenal SinhaAlesssandro Arduini
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
Our protocol describes a simple procedure for isolating stromal cells from lymph nodes (LN). LN are disrupted then enzymatically digested with collagenase and dispase to produce a single cell suspension that can be stained with fluorescently labelled antibodies and analysed by flow cytometry. This protocol will enable identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC) as PNAd+ BEC that form LN high endothelial venules (HEV). This method can be applied to examine LN stromal cell responses during inflammatory events induced by infections or immunologic adjuvants and to subset most leukocytes found in LN.
Keywords: Lymphoid stromal cells Lymph nodes Endothelial cells Fibroblastic reticular cells Immune response Virus infection
Background
Lymph nodes (LN) are constructed of complex networks of mesenchymal and endothelial stromal cells. These include the fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs) and blood endothelial cells (BECs). These stromal cells organize the complex microarchitecture of LN, enabling the support of immune cell migration, homeostasis, tolerance and cellular interactions required for the initiation of immune responses to pathogens and tumors. We have shown that the LN stromal cells can proliferate and expand in response to inflammatory signals and the recruitment of immune cells into LN that accompanies infections (Gregory et al., 2017). These stromal cells can also significantly modulate their transcriptional program to respond to infection, thereby supporting ongoing immune responses. This protocol enables reliable isolation of stromal cell subsets from LN both in the steady state and during disease. This enables phenotypic, functional, genetic or epigenetic investigation of LN stromal cells to reveal how they contribute to tissue homeostasis and immune responses.
Materials and Reagents
1,000 μl pipette tips (Neptune, catalog number: BT1250 )
200 μl pipette tips (Neptune, catalog number: BT200 )
20 μl pipette tips (Neptune, catalog number: BT20 )
10 μl pipette tips (Neptune, catalog number: BT10XLS3 )
24-well plate (Corning, catalog number: 3527 )
10 ml tubes (SARSTEDT, catalog number: 62.9924.284 )
50 ml tubes (Greiner Bio One International, catalog number: 227261 )
Nylon mesh 70 μm (Clear Edge, catalog number: PA75-1750 )
5 ml polystyrene round bottom tube (Corning, Falcon®, catalog number: 352008 )
96-well round-bottom plate (Corning, catalog number: 3788 )
Spatula (VWR, catalog number: 10806-408 )
C57BL\6 mice (THE JACKSON LABORATORY, catalog number: 000664 )
Blank calibration particles (BD, BD Biosciences, catalog number: 556296 )
HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 11344033 )
L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21051024 )
Benzylpenicillin (CSL, catalog number: AUST R 10329 )
Streptomycin sulphate (Thermo Fisher Scientific, GibcoTM, catalog number: 11860038 )
2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
RPMI (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
Collagenase D (Sigma-Aldrich, catalog number: 11088882001 )
Manufacturer: Roche Diagnostics, catalog number: 11088882001 .
Dispase II (Sigma-Aldrich, catalog number: 04942078001 )
Manufacturer: Roche Diagnostics, catalog number: 04942078001 .
DNase I (Sigma-Aldrich, catalog number: DN25-1G )
Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906-500G )
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134-1KG )
Antibodies
gp38-PE, 8.1.1 (BioLgend, catalog number: 127408 )
CD31-PE-Cy7, 390 (BioLegend, catalog number: 102524 )
CD45.2-A700, 104 (BioLegend, catalog number: 109822 )
Ter-119-A700, TER-119 (BioLegend, catalog number: 116220 )
HEV-A488, MECA-79 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 53-6036-82 )
CD16/32-purified, 2.4G2 (BD, BD Biosciences, catalog number: 553142 )
Live/Dead Fixable Near-IR cell stain kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: L10119 )
Supplementum Completum (SC) (see Recipes)
RP-2 solution (see Recipes)
Digestion mix 1 (see Recipes)
Digestion mix 2 (see Recipes)
FACS buffer (see Recipes)
Lymph node stromal cell antibody cocktail (see Recipes)
Equipment
Water bath (VWR, catalog number: 89501-464 )
Dumont No.5 forceps (Fine Science Tools, catalog number: 11251-10 )
Graefe forceps (Roboz Surgical Instrument, catalog number: RS-5137 )
Analytical balance for weighing (Ohaus, catalog number: 30208454 )
1,000 μl pipette (Eppendorf, catalog number: 3121000120 )
200 μl pipette (Eppendorf, catalog number: 3121000082 )
20 μl pipette (Eppendorf, catalog number: 3121000040 )
10 μl pipette (Eppendorf, catalog number: 3121000015 )
Refrigerated centrifuge with plate carriers (Beckman Coulter, model: Allegra® X-15R , catalog number: B09822; Beckman Coulter, catalog number: 368914 )
Flow cytometer (BD, BD Biosciences, model: BD LSRFORTESSA )
Software
FlowJo (FlowJo)
Prism (GraphPad)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Alexandre, Y. O. and Mueller, S. N. (2017). Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes. Bio-protocol 7(16): e2445. DOI: 10.21769/BioProtoc.2445.
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Cell Biology > Cell isolation and culture > Cell isolation
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2,446 | https://bio-protocol.org/exchange/protocoldetail?id=2446&type=0 | # Bio-Protocol Content
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Peer-reviewed
GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression
ZL Zhao Liu
WW Wei Wang
CZ Chun-Guang Zhang
JZ Jun-Feng Zhao
Y Yu-Ling Chen
Published: Vol 7, Iss 14, Jul 20, 2017
DOI: 10.21769/BioProtoc.2446 Views: 16801
Edited by: Scott A M McAdam
Reviewed by: Honghong Wu
Original Research Article:
The authors used this protocol in Jun 2017
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Abstract
Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll and guard cells, it is hard to exhibit a clear expression of the gene in guard cells by a GUS staining image from leaf. To gain a clear guard cell GUS image of small G protein ROP7, a gene expressed in both mesophyll and guard cells, we peeled the epidermal strips from the leaf of 3-4 week-old plants. After removing the mesophyll cells, the epidermal strips were used for GUS staining. We compared the GUS staining images from epidermal strips or leaf of small G protein ROP7 and RopGEF4, a gene specifically expressed in guard cells, and found that GUS staining of epidermal strips provided a good method to show the guard cell expression of a gene expressed in both mesophyll and guard cells. This protocol is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.
Keywords: Guard cells Gene expression GUS staining Epidermal strip Leaf
Background
Stomatal movements regulate the gas exchange between plants and environment, therefore, it is important to reveal the mechanism of the opening or closure of stomata. Determination of the guard cell expression of a gene is essential for studying its role in stomatal movements. There are several ways to identify the expression of a gene in guard cells. One way is to check the RNA expression of a gene in guard cells by RT-PCR (Jeon et al., 2008; Takimiya et al., 2013). To do so, the protoplasts of mesophyll and guard cells need to be separated. Another way is to check the GUS signal in guard cells of the transgenic plant expressing GUS driven by a gene’s native promoter. In some reports, the evidence of both RNA expression and GUS signal in guard cells were provided (Zheng et al., 2002; Jeon et al., 2008). As for the GUS signal in guard cells, if a gene is specifically expressed in guard cells, like OST1, MYB60, ROP11 and RopGEF4, a distinguished GUS signal in guard cells can be obtained from a GUS staining image with whole leaf (Mustilli et al., 2002; Li et al., 2012; Li and Liu, 2012; Rusconi et al., 2013). However, if a gene is expressed in both mesophyll and guard cells, like ROP10 and RopGEF2, the GUS signal in guard cells is hard to be distinguished from the mesophyll background (Zheng et al., 2002; Li and Liu, 2012). After GUS staining procedure, the leaf will become soft, and it is very difficult to peel the epidermal strips. Therefore, just after the leaf was excised from the plants, we peeled the epidermal strips from the leaf, and the strips were used for GUS staining after the mesophyll cells were removed. By this method, we obtained a clear guard cell GUS image of ROP7, a gene expressed in both mesophyll and guard cells.
Materials and Reagents
Pipette tips
1.5 ml Eppendorf tubes
Sterilized filter paper
Plastic Petri dishes for plant culture
Slide
Cover glass
0.45 micron filter
Aluminum foil
Arabidopsis thaliana seeds of ROP7pro:GUS and RopGEF4pro:GUS lines
100%, 75%, 40%, 20%, 10%, 5% ethanol in water
50% glycerol (Sangon Biotech, catalog number: A100854 )
100% methanol
37% hydrochloric acid (12 N)
Sodium hydroxide (AMRESCO, catalog number: 0583 )
Ethylenediaminetetraacetate acid (EDTA) (AMRESCO, catalog number: 0322 )
Triton X-100 (AMRESCO, catalog number: 0694 )
Potassium ferricyanide (AMRESCO, catalog number: 0713 )
Potassium ferrocyanide (Sigma-Aldrich, catalog number: P9387 )
X-Gluc (Sigma-Aldrich, catalog number: B5285 )
Dimethylformamide (AMRESCO, catalog number: 0464 )
Sodium dihydrogen phosphate (NaH2PO4·H2O) (Sigma-Aldrich, catalog number: S9638 )
Disodium hydrogen phosphate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: 431478 )
20% methanol in 0.24 N hydrochloric acid (see Recipes)
60% ethanol in 7% sodium hydroxide (see Recipes)
GUS staining solution (see Recipes)
Equipment
Pipetman 100 μl (Gilson, catalog number: F123615 )
Pipetman 1,000 μl (Gilson, catalog number: F123602 )
Tweezers
Brush pen
Plant growth chamber (Percival Scientific, model: CU-36L5 ) and greenhouse
Pots
Incubator at 37 °C (SANFA, model: DNP-9052 )
Microscope (ZEISS, model: Axio Imager A1 )
Water purification system (deionized water) (EMD Millipore, model: Elix® Essential , 5 L)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Liu, Z., Wang, W., Zhang, C., Zhao, J. and Chen, Y. (2017). GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression. Bio-protocol 7(14): e2446. DOI: 10.21769/BioProtoc.2446.
Wang, W., Liu, Z., Bao, L. J., Zhang, S. S., Zhang, C. G., Li, X., Li, H. X., Zhang, X. L., Bones, A. M., Yang, Z. B. and Chen, Y. L. (2017). The RopGEF2-ROP7/ROP2 Pathway Activated by phyB Suppresses Red Light-Induced Stomatal Opening. Plant Physiol 174(2): 717-731.
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Category
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > Gene expression
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2,447 | https://bio-protocol.org/exchange/protocoldetail?id=2447&type=0 | # Bio-Protocol Content
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Isolation of Guard-cell Enriched Tissue for RNA Extraction
PJ Pirko Jalakas
Dmitry Yarmolinsky
Hannes Kollist
Mikael Brosche
Published: Vol 7, Iss 15, Aug 5, 2017
DOI: 10.21769/BioProtoc.2447 Views: 15257
Edited by: Scott A M McAdam
Reviewed by: Yi Zhang
Original Research Article:
The authors used this protocol in Jun 2017
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Abstract
This is a protocol for isolation of guard cell enriched samples from Arabidopsis thaliana plants for RNA extraction. Leaves are blended in ice-water and filtered through nylon mesh to obtain guard cell enriched fragments. With guard cell enriched samples, gene expression analysis can be done, e.g., comparing different gene expression levels in guard cells versus whole leaf to determine if a gene of interest is predominantly expressed in guard cells. It can also be used to study the effect of treatments or different genetic backgrounds in the regulation of the guard cell expressed genes.
Keywords: Arabidopsis thaliana Guard cell isolation RNA
Background
Isolation of guard cells for RNA extraction has traditionally relied on guard cell protoplast extraction (Leonhardt et al., 2004) or epidermal peels (Pandey et al., 2010). Manual dissection of guard cells from freeze-dried leaves has also been used (Bates et al., 2012). The protoplast procedure may introduce unwanted changes in gene expression from wounding effects and other methods are time consuming, require special equipment or expensive reagents (e.g., transcriptional inhibitors). Hence there is a need for a fast and simple protocol to isolate guard cells for gene expression analysis. Here we further describe a quick ice blender method for isolation of guard cell enriched tissue (Bauer et al., 2013), in the form of epidermal fragments without mesophyll and other vascular cells. This method uses a nylon mesh to collect epidermis from blended leaf tissue. The adequately large pore size of the nylon mesh allows mesophyll and other vascular cells to pass through while retaining the epidermal fragments. We propose that the critical factor in this protocol is the type of blender used to process the samples.
Materials and Reagents
Razor blade
210 μm nylon mesh (A. Hartenstein, laborversand.de, catalog number: PAS1 )
Plastic embroidery hoop from a handicraft store (Figure 1)
Note: Nylon mesh is placed in the plastic embroidery hoop to provide a little tension to the mesh, to make it easier to pour the blended water and guard cell enriched fragments through the mesh.
Figure 1. Nylon mesh with a plastic embroidery hoop
Paper towel
Small medical spatula or plastic spoon
1.5 ml tube or aluminum foil
Arabidopsis plants
Milli-Q or distilled water kept at 4 °C
Crushed ice
Liquid nitrogen
Equipment
Blender (Braun JB 3060 or other 800 W blender) (Braun Household, model: JB 3060 )
Note: A blender with lower effect has been tried, but failed to isolate proper fragments, probably due to insufficient power to fully crush the ice.
Microscope (ZEISS, model: SteREO Discovery.v20 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Jalakas, P., Yarmolinsky, D., Kollist, H. and Brosche, M. (2017). Isolation of Guard-cell Enriched Tissue for RNA Extraction. Bio-protocol 7(15): e2447. DOI: 10.21769/BioProtoc.2447.
Jalakas, P., Huang, Y.-C., Yeh, Y.-H., Zimmerli, L., Merilo, E., Kollist, H. and Brosché, M. (2017). The role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis stomatal responses is beyond ABA signaling. Plant Physiol 174: 665-671.
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Category
Plant Science > Plant cell biology > Cell isolation
Cell Biology > Cell isolation and culture > Cell isolation
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2,448 | https://bio-protocol.org/exchange/protocoldetail?id=2448&type=0 | # Bio-Protocol Content
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Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants
KR Karen Sue Renzaglia
Renee A. Lopez
JH Jason S. Henry
NF Nicholas D. Flowers
KV Kevin C. Vaughn
Published: Vol 7, Iss 19, Oct 5, 2017
DOI: 10.21769/BioProtoc.2448 Views: 8440
Edited by: Scott A M McAdam
Original Research Article:
The authors used this protocol in Jun 2017
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Abstract
Motile male gametes (spermatozoids) of land plants are coiled and contain a modified and precisely organized complement of organelles that includes a locomotory apparatus with two to thousands of flagella. Each flagellum is generated from a basal body that originates de novo as a centriole in spermatogenous cell lineages. Much of what is known about the diversity of plant male gametes was derived from detailed transmission electron microscopic studies. Because the process of spermatogenesis results in complete transformation of the shape and organization of these cells, TEM studies have yielded a wealth of information on cellular differentiation. Because green algal progenitor groups contain centrioles and a variety of motile cells, land plant spermatozoids also provide a plethora of opportunities to examine the evolution and eventual loss of centrioles and locomotory apparatus during land colonization.
Here we provide a brief overview of the studies and methodologies we have conducted over the past 20 years that have elucidated not only the structural diversity of these cells but also the development of microtubule organizing centers, the de novo origin of centrioles and the ontogeny of structurally complex motile cells.
Keywords: Blepharoplast Basal bodies Centrioles Extracellular matrix Flagella Transmission electron microscopy
Background
Motile gametes of land plants are strikingly diverse and develop through transformations that involve repositioning, and reshaping of cellular components, and the assembly of a complex locomotory apparatus (Renzaglia and Garbary, 2001; Lopez and Renzaglia, 2008). Because of constraints imposed by cell walls, elongation of the cell and flagella is around the periphery of a nearly spherical space, resulting in a coiled configuration of the mature gamete (Renzaglia and Garbary, 2001; Lopez and Renzaglia, 2014). The degree of coiling varies from just over one to as many as 10 revolutions per cell. The number of flagella per gamete is even more variable, ranging from two in bryophytes (mosses, hornworts, liverworts and most lycophytes) to an estimated 1,000-40,000 in Ginkgo and cycads, the earliest divergent seed plant lineages. Following the diversification of Ginkgo and cycads, all vestiges of basal bodies and flagella were lost in the remaining seed plants that utilize pollen tubes to deliver non-motile sperm to egg cells (Southworth and Cresti, 1997).
It is widely known that vegetative plant cells lack centrioles and the centrosome is elusive. A lesser-known fact is that in plants with motile sperm cells, centrioles arise de novo during the penultimate or ultimate mitotic divisions that produce the nascent spermatid in antheridia (Renzaglia and Carothers, 1986; Vaughn and Renzaglia, 1998; Vaughn and Harper, 1998; Renzaglia and Maden, 2000; Vaughn and Renzaglia, 2006). In these cell lineages, centriolar centrosomes serve as the nucleation site for spindle microtubules and thus bear striking parallels with centrioles of animal and protist cells. In the developing sperm cells, the centrioles reposition, anchor to form the distinctive basal bodies, and elongate to produce the 2-40,000 flagella in each gamete. These changes occur in synchrony with cell elongation, and the entire process of cytomorphogenesis is guided by the production of unique arrays of microtubules, and fibrillar and lamellar bands or strips. Because of the exclusive occurrence of basal bodies, flagella and associated complexes in developing male gametes, studies of spermatogenesis have revealed important information on the structure, composition, and developmental changes in microtubule arrays as they relate to the cell cycle, microtubule organizing centers (MTOCs), and cellular differentiation in plants. The purpose of this review is to describe the method used in transmission electron microscopic examination and to demonstrate how this approach has advanced understanding of basal bodies, flagella/cilia, and associated structures in land plants.
Materials and Reagents
Transmission electron microscope (TEM)
Scintillation vials with aluminum covered caps (Fisher Scientific, catalog number: 03-340-4B)
Manufacturer: DWK Life Sciences, Kimble®, catalog number: 7450320 .
BEEM embedding capsules size ‘00’ (Electron Microscopy Sciences, catalog number: 70000-B )
Formfar Carbon Film 200 mesh Ni grids (Electron Microscopy Sciences, catalog number: FCF200-Ni )
Copper 200 mesh grids (Electron Microscopy Sciences, catalog number: EMS200-Cu )
Sperm cells
Megaceros flagellaris
Phaeoceros carolinianus
Phylloglossum drummondii
Ginkgo biloba
Angiopteris evecta
Conocephalum conicum
Ceratopteris richardii
Riccardia multifida
Aulacomnium palustre
Equisetum arvense
Ethanol (Decon Labs, catalog number: 2705HC )
Low viscosity resin
Glutaraldehyde (Electron Microscopy Sciences , catalog number: 16120 )
Sorensens phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences , catalog number: 11600-10 )
Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
Potassium ferrocyanide (Fisher Scientific, catalog number: P236-500 )
Uranyl acetate (Polyscience, catalog number: 21447-25 )
100% methanol
Lead nitrate (Electron Microscopy Sciences, catalog number: 17900 )
Sodium citrate (Electron Microscopy Sciences, catalog number: 21140 )
1 N NaOH
100% propylene oxide (Electron Microscopy Sciences, catalog number: 20401 )
2.5% glutaraldehyde (see Recipes)
0.05 M phosphate buffer (pH 7.2) (see Recipes)
4% aqueous osmium tetroxide (see Recipes)
Equipment
Transmission electron microscope (TEM)
Diamond knife (Diatome, specs: Ultra, 45°, 4 mm, Wet)
Transmission electron microscope (Hitachi, model: HF7100 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Renzaglia, K. S., Lopez, R. A., Henry, J. S., Flowers, N. D. and Vaughn, K. C. (2017). Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants. Bio-protocol 7(19): e2448. DOI: 10.21769/BioProtoc.2448.
Renzaglia, K. S., Villarreal, J. C., Piatkowski, B. T., Lucas, J. R. and Merced, A. (2017). Hornwort Stomata: Architecture and Fate Shared with 400-Million-Year-Old Fossil Plants without Leaves. Plant Physiol 174(2): 788-797.
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Category
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Electron microscopy
Cell Biology > Cell movement > Cell motility
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2,449 | https://bio-protocol.org/exchange/protocoldetail?id=2449&type=0 | # Bio-Protocol Content
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Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density
HW Hui-Chen Wu
YH Ya-Chen Huang
CL Chia-Hung Liu
Tsung-Luo Jinn
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2449 Views: 9311
Edited by: Scott A M McAdam
Reviewed by: Joëlle SchlapferAlizée Malnoe
Original Research Article:
The authors used this protocol in May 2017
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Abstract
The number of stomata on leaves can be affected by intrinsic development programming and various environmental factors, in addition the control of stomatal apertures is extremely important for the plant stress response. In response to elevated temperatures, transpiration occurs through the stomatal apertures, allowing the leaf to cool through water evaporation. As such, monitoring of stomata behavior to elevated temperatures remains as an important area of research. The protocol allows analysis of stomatal aperture, morphology, and density through a non-destructive imprint of Arabidopsis thaliana leaf surface. Stomatal counts were performed and observed under a scanning electron microscope.
Keywords: Arabidopsis thaliana Heat stress Non-destructive imprint Stomata Scanning electron microscope
Background
The different techniques have been explored to study stomatal density and patterns in variety of plants. It can be broadly grouped into two classes including direct observation of fresh materials and the preparation of replicas, or castings (Gitz and Baker, 2009). These methods have included the use of clear nail varnish, which is a traditional method used to analyze stomata density, however, surfaces of leaves can be damaged by the solvent in the nail varnish. Here we use a non-destructive imprint of Arabidopsis thaliana leaves for stomata visualization, which can directly observe freshly collected materials and assess stomata density for sequential measurements. Additionally, it can simplify the observation and the measurement of stomatal density, and facilitate the analysis of several lines or different treatments in parallel.
Materials and Reagents
Arabidopsis thaliana
Glass slide
Lens cleaning tissue (GE Healthcare, Whatman, catalog number: 2105-862 )
Toothpick
Double-sided tape
Dental silicon (vinyl polysiloxane; VPS) impression materials: EXAFINE VPS impression material injection type, with low viscosity (GC America, catalog number: 138120 )
Note: Two components–base (A) and catalyst (B).
High strength 5-min Epoxy gel (ITW Devcon, catalog number: 14210 )
Note: Two components–resin (C) and hardener (D).
Ultrafine threads used in ophthalmologic surgery (Fine Science Tools, catalog number: 18020-03 )
Equipment
Forceps (Fine Science Tools, catalog number: 00108-11 )
Forced convection oven (YIHDER Technology, model: DK-600D )
Stereo microscope (ZEISS, model: Stemi DV4 )
Scanning electron microscopy (SEM; Inspect S, FEI)
Sputtering coater (EIKO Engineering, model: IB-2 )
Software
ImageJ software (http://rsb.info.nih.gov/ij)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Wu, H., Huang, Y., Liu, C. and Jinn, T. (2017). Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density. Bio-protocol 7(16): e2449. DOI: 10.21769/BioProtoc.2449.
Huang, Y. C., Wu, H. C., Wang, Y. D., Liu, C. H., Lin, C. C., Luo, D. L. and Jinn, T. L. (2017). PECTIN METHYLESTERASE34 contributes to heat tolerance through its role in promoting stomatal movement. Plant Physiol 174: 748-763.
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Category
Plant Science > Plant cell biology > Tissue analysis
Cell Biology > Cell imaging > Fixed-tissue imaging
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245 | https://bio-protocol.org/exchange/protocoldetail?id=245&type=0 | # Bio-Protocol Content
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Tandem Affinity Purification in Drosophila Heads and Ovaries
AP Anita Pepper
BB Balpreet Bhogal
TJ Thomas Jongens
Published: Vol 2, Iss 15, Aug 5, 2012
DOI: 10.21769/BioProtoc.245 Views: 13505
Original Research Article:
The authors used this protocol in Dec 2011
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Abstract
Tandem affinity purification (TAP) (Pugi et al.,2001; Rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three components: a calmodulin-binding peptide, a Tobacco etch virus (TEV) protease cleavage site and Protein A which is an immunoglobulin G (IgG)-binding domain. This protocol was modified from the original methodology used in yeast cells(Pugi et al.,2001; Rigaut et al., 1999) for isolation of protein complexes from Drosophila heads and ovaries expressing a TAP tagged protein of interest. To determine in vivo binding partners of the Drosophila fragile X protein (dFMR1), we developed a transgenic strain of flies expressing a recombinant form of dFMR1 with a carboxy-terminal TAP tag (Tsai and Carstens, 2006). To ensure that the construct was expressed at wild-type levels, we engineered this form of the tagged protein in the context of a genomic rescue construct that rescued a mutant sterility phenotype. The purification process was performed using mild conditions to maintain native protein interactions. For TAP methods in Drosophila S2 cell culture, we have successfully used a protocol previously published by Tsai and Carstens (Tsai and Carstens, 2006; Bhogal et al., 2011).
Materials and Reagents
1x Phosphate buffered saline (PBS)
Fly sieves: 106, 355, 600 and 850 mm
IgG Sepharose Fast Flow Beads (GE Healthcare Dharmacon, catalog number: 17-0969-01 )
AcTEV Protease (Life Technologies, Invitrogen™, catalog number: 12575-015 )
Calmodulin Sepharose 4B (GE Healthcare Dharmacon, catalog number: 17-0529-01 )
Micro Bio-Spin column (Bio-Rad Laboratories, catalog number: 732-6204 )
NuPAGE Novex 4-12% Bis-Tris gel (Life Technologies, catalog number: NP0321 )
Silverquest Silver Staining Kit (Life Technologies, catalog number: LC6070 ), Colloidal Blue Staining Kit (Life Technologies, catalog number: LC6025 ) or Coomassie Blue.
Beta-mercaptoethanol
1 M CaCl2
Hepes
MgCl2
KCl
NP-40
DTT
Na3VO4
EDTA
EGTA
NaF
Glycerol
Mg-acetate
Imidazole
Bouwmeester's buffer (see Recipes)
Buffer A (see Recipes)
Buffer B (see Recipes)
Buffer C (see Recipes)
TEV Cleavage buffer (see Recipes)
Calmodulin binding buffer (see Recipes)
Calmodulin blution buffer (see Recipes)
Equipment
Benchtop centrifuge (that fits 1.5 ml microcentrifuge tubes)
1.5 ml microcentrifuge tubes
Nutator
Vortexer
Ceramic mortar and pestle
Dounce homogenizer
Disposable pestles for 1.5 ml tubes
Beckman table top centrifuge (that fits 15 ml and 50 ml conical tubes and can be cooled to 4 °C)
Sorvall with a Beckman JA-20 rotor that is capable to cool to 4 °C
Mass Spectrometry Facility
Procedure
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Category
Developmental Biology > Cell signaling > TAP tag
Biochemistry > Protein > Interaction
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2,450 | https://bio-protocol.org/exchange/protocoldetail?id=2450&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Cell-free Generation of COPII-coated Procollagen I Carriers
LY Lin Yuan
SB Satoshi Baba
KB Kanika Bajaj
RS Randy Schekman
Published: Vol 7, Iss 22, Nov 20, 2017
DOI: 10.21769/BioProtoc.2450 Views: 7355
Edited by: Gal Haimovich
Original Research Article:
The authors used this protocol in Jun 2017
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Jun 2017
Abstract
The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the isolation and detection of reconstituted COPII-coated carriers. This cell-free reaction can be used to test conditions that stimulate or suppress the packaging of PC1 into COPII-coated carriers.
Keywords: COPII Collagen Membrane Budding Reconstitution
Background
The coat protein complex II (COPII) plays an essential role in transporting secretory cargos from the endoplasmic reticulum (ER) en route to the Golgi apparatus. The genes required for cargo traffic from the ER were discovered in genetic studies in yeast and the precise roles of the protein products of the genes required for vesicle budding were elucidated with the aid of a cell-free vesicle budding reaction supplemented with purified components (Novick et al., 1981; Kaiser et al., 1990; Barlowe et al., 1994). A similar reaction was developed to detect the role of COPII in cargo traffic from the ER in cultured mammalian cells (Kim et al., 2005). Mammalian COPII-coated vesicles are approximately 80-100 nm in diameter, which is seemingly too small to accommodate large secretory cargos such as the rigid 300 nm procollagen I (PC1) triple helical rod. Despite the potential size discrepancy, COPII is essential for the secretion of large cargos including PC1 (Boyadjiev et al., 2006). Recently, we reported the existence of bona fide large COPII-coated PC1 carriers, exceeding 300 nm in diameter, in cells evaluated by stochastic optical reconstruction microscopy (STORM), correlated light electron microscopy (CLEM) and live-cell imaging (Gorur et al., 2017). Cell-free COPII budding reactions that successfully reconstituted small COPII vesicles did not allow the detection of large COPII-coated PC1 carriers (Fromme and Schekman, 2005). Therefore, we devised an alternative vesicle budding protocol to allow the detection of PC1 packaged into large COPII vesicles as well as the characterization of both small and large COPII-coated vesicles. Using this new protocol, we showed that the capture of PC1 into large COPII vesicles requires COPII proteins and the GTPase activity of the COPII subunit SAR1 (Gorur et al., 2017).
Materials and Reagents
Falcon® 150 mm TC-treated cell culture dish (Corning, Falcon®, catalog number: 353025 ) or equivalent
BioExpress GeneMate 50 ml centrifuge tubes (BioExpress, Greiner Bio One, catalog number: C-3394-4 ) or equivalent
BioExpress GeneMate racked pipet tips, low retention, 200 μl (BioExpress, catalog number: P-1234-200)
Manufacturer: Biotix, catalog number: P-1234-200CS .
BioExpress GeneMate racked pipet tips, low retention, 1,000 μl (BioExpress, catalog number: P-1234-1000)
Manufacturer: Biotix, catalog number: P-1234-1000CS .
BioExpress GeneMate 15 ml centrifuge tubes (BioExpress, Greiner Bio One, catalog number: C-3394-2 ) or equivalent
Falcon® 100 mm TC-treated cell culture dish (Corning, Falcon®, catalog number: 353003 ) or equivalent
Amicon® Ultra-15 ml centrifugal filter unit with Ultracel-3K membrane (Merck, catalog number: UFC900324 )
Amicon® Ultra-0.5 ml centrifugal filter unit with Ultracel-3K membrane (Merck, catalog number: UFC500324 )
Oxygen® 1.5 ml MAXYMmum recoveryTM microcentrifuge tube (low retention) (Corning, Axygen®, catalog number: MCT-150-L-C )
Microscope slides (Fisher Scientific, catalog number: 12-550-343 ) or equivalent
Microscope cover glass (Fisher Scientific, catalog number: 12-542A ) or equivalent
Tube, 7 x 20 mm, thickwall, polycarbonate (Beckman Coulter, catalog number: 343775 )
Prot/Elec tips (gel loading tips) (Bio-Rad Laboratories, catalog number: 2239915 )
Cell scraper 25 cm (SARSTEDT, catalog number: 83.1830 ) or equivalent
Corning 1 L filter system 0.22 μm (Corning, catalog number: 431098 ) or equivalent
Steriflip® 50 ml filter 0.22 μm (Merck, catalog number: SCGP00525 ) or equivalent
Posi-click 1.7 ml micro-centrifuge tube (Danville Scientific, catalog number: C2170 (1001002)) or equivalent
Microfuge tube, polypropylene, 1.5 ml (Beckman Coulter, catalog number: 357448 )
Cuvettes (SARSTEDT, catalog number: 67.742 )
Immobilon®-P transfer membrane PVDF 0.45 μm (Merck, catalog number: IPVH00010 )
HT-1080 human fibrosarcoma (ATCC, catalog number: CCL-121 ) for cytosol preparation
Note: Other fast-growing cell lines that support PC1 secretion may also be used for this purpose.
IMR-90 human lung fibroblasts (Coriell Cell Repositories at the National Institute on Aging, Coriell Institute for Medical Research) (Coriell Institute, catalog number: I90-83 ) for donor membrane preparation
Note: Other cell lines that express endogenous PC1 and prolific at PC1 secretion may be used for this purpose. For this reaction, it is important to use young IMR-90 with cumulative Population Doubling Level (PDL) lower than 37.5, because aged cells secrete significantly less PC1. PDL was calculated using a standard formula: cumulative PDL = initial PDL + 3.32 [log (current cell yield) - log (cell plated)].
Phosphate-buffered saline (PBS, pH 7.4)
cOmpleteTM, EDTA-free, protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 05056489001 )
Bio-beadsTM SM-2 adsorbent media (Bio-Rad Laboratories, catalog number: 1523920 )
Bio-Rad protein assay dye reagent concentrate (Bradford) (Bio-Rad Laboratories, catalog number: 5000006 )
Liquid nitrogen
0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
HyClone® trypan blue solution (GE Healthcare, HyCloneTM, catalog number: SV30084 )
OptiPrepTM density gradient medium (Sigma-Aldrich, catalog number: D1556 )
NovexTM WedgeWellTM 4-20% Tris-glycine gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: XP04205BOX )
Antibodies
Rabbit anti-PC1 (LF-41) was a gift from L. Fisher (National Institute of Dental and Craniofacial Research, Bethesda, MD), and it was used at 1:5,000
Rabbit anti ribophorin I, ERGIC53, and SEC22B were made in-house and they were used at 1:5,000
Mouse anti HSP47 (Enzo Life Sciences, catalog number: ADI-SPA-470-D ), and it was used at 1:5,000
PierceTM ECL 2 Western blotting substrate (Thermo Fisher Scientific, catalog number: 32132 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3294-100G )
Life Science Seradigm premium grade fetal bovine serum (FBS) (VWR, catalog number: 1500-500 )
DMEM, GlutaMAXTM (Thermo Fisher Scientific, GibcoTM, catalog number: 10566016 )
HEPES (Sigma-Aldrich, catalog number: RDD002-1KG )
Potassium hydroxide (KOH)
D-Sorbitol (Sigma-Aldrich, catalog number: S1876-5KG )
Potassium acetate (KoAc) (Fisher Scientific, catalog number: BP364-500 )
Magnesium acetate tetrahydrate (MgoAc) (Sigma-Aldrich, catalog number: M0631-500G )
Sodium dodecyl sulfate (SDS) (Avantor Performance Materials, J.T.Baker®, catalog number: 4095-02 )
Glycerol (AMRESCO, catalog number: M152-4L )
Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
Glycine (Fisher Scientific, catalog number: BP381-5 )
2-Mercaptoethanol (βME) (AMRESCO, catalog number: M131-100ML )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
Tris base (Fisher Scientific, catalog number: BP152-5 )
Triton® X-100 (Sigma-Aldrich, catalog number: X100-500ML )
TWEEN® 20 (Sigma-Aldrich, catalog number: P7949-500ML )
Tris-buffered saline (TBS, pH 7.6)
Digitonin (Sigma-Aldrich, catalog number: D141-500MG )
Dimethyl sulfoxide (DSMO) (Sigma-Aldrich, catalog number: D8418-100ML )
Trypsin inhibitor from glycine max (soybean) (Sigma-Aldrich, catalog number: T9003 )
Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: 203637 )
Creatine phosphate (Sigma-Aldrich, catalog number: 2380-25GM )
Creatine kinase (Roche Diagnostics, catalog number: 10127566001 )
Adenosine 5’-triphosphate (ATP) (GE Healthcare, catalog number: 27-1006-01 )
GTP 100 mM Li Salt (Sigma-Aldrich, Roche Diagnostics, catalog number: 11140957001 )
Methanol (Fisher Scientific, catalog number: A452-4 )
Mammalian cell culture medium (see Recipes)
Buffer solutions (see Recipes)
B88
B88-0
Sample buffer (5x)
Buffer C
Sample buffer C (1x)
Transfer buffer
HK buffer
TBST
Stock solutions (see Recipes)
1 M HEPES pH 7.2
10% SDS
Digitonin stock
Trypsin inhibitor stock
0.5 M LiCl
ATP regeneration system (ATP r.s.)
GTP
Equipment
SorvallTM ST16R centrifuge, TX-200 Swinging Bucket Rotor, 400 ml Round Buckets, 4 x 50 ml, 9 x 15 ml conical adapters (Thermo Fisher Scientific, model: SorvallTM ST 16R , catalog number: 75818382) or equivalent
TLA-55 ultracentrifuge rotor (Beckman Coulter, model: TLA-55 , catalog number: 366725)
Centrifuge 5430R, refrigerated with fixed angle rotor FA-45-30-11 (Eppendorf, model: 5430 R , catalog number: 5428000015)
Light microscope with a 16x or 25x objective (any simple or compound light microscope is fine)
S-24-11-AT swinging bucket rotor (Eppendorf, catalog number: 5409715003 )
Spectronic Genesys 5 spectrophotometer (Spectronic Instruments) or equivalent
Table top ultracentrifuge, we used Optima MAX-XP, Optima TL, and Optima TL-100 for this experiment (Beckman Coulter, models: OptimatTM MAX-XP , OptimaTM TL , OptimaTM TL-100 )
Microman® positive-displacement pipet M250 (Gilson, catalog number: F148505 )
Microman® capillary pistons for M250 (Gilson, catalog number: F148114 )
TLS-55 swinging bucket ultracentrifuge rotor (Beckman Coulter, model: TLS-55 , catalog number: 346936)
TLS-55 adapter, Delrin, for 7 x 20 mm tubes (Beckman Coulter, catalog number: 358615 )
Micro tube mixer MT-360 (TOMY SEIKO, model: MT-360 )
BioExpress GeneMate GyroMixer XL (BioExpress, GeneMate, catalog number: R-3200-1XL ) or equivalent platform rotator
ChemiDocTM MP Imaging System (Bio-Rad Laboratories, model: ChemiDocTM MP )
Small beaker
Software
ImageLab software v4.0
ImageJ
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Yuan, L., Baba, S., Bajaj, K. and Schekman, R. (2017). Cell-free Generation of COPII-coated Procollagen I Carriers. Bio-protocol 7(22): e2450. DOI: 10.21769/BioProtoc.2450.
Gorur, A., Yuan, L., Kenny, S. J., Baba, S., Xu, K. and Schekman, R. (2017). COPII-coated membranes function as transport carriers of intracellular procollagen I. J Cell Biol 216(6): 1745-1759.
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Category
Biochemistry > Lipid > Lipid-protein interaction
Biochemistry > Protein > Isolation and purification
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2,451 | https://bio-protocol.org/exchange/protocoldetail?id=2451&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Capturing Z-stacked Confocal Images of Living Bacteria Entering Hydathode Pores of Cauliflower
AC Aude Cerutti
Alain Jauneau
Published: Vol 7, Iss 20, Oct 20, 2017
DOI: 10.21769/BioProtoc.2451 Views: 8204
Original Research Article:
The authors used this protocol in Jun 2017
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Jun 2017
Abstract
The present protocol to visualize living bacteria at the pore level of cauliflower hydathodes is simple and trained users in confocal microscopy can execute it successfully. It can be easily adapted to capture images with other plant-microorganism interactions at the leaf surface and should be useful to obtain important information on pore and stomatal biology. A critical limitation to methods used to observe plant-microorganism interactions in the pore is the application of too much pressure to the sample during observations and z-stack acquisitions. To solve this issue, we recommend the use of a long working-distance water immersion objective lens that allows observations even with thick samples.
Keywords: Cauliflower Pore Hydathode Xanthomonas Confocal microscope
Background
Pores of hydathodes and stomata are possible entry points for pathogenic microorganisms to invade plant tissues. In cauliflower, the hydathodes present on leaf margins exhibit large pores, resembling stomata. These pores are routes for the leaf infection by the vascular pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) (Cerutti et al., 2017). Hydathodes are present on leaves of a wide range of vascular plants. We describe a simple protocol to visualize bacteria at the pore level by confocal microscopy.
Materials and Reagents
Razor blade
Microscope glass slides (Thermo Fisher Scientific, Superfrost, catalog number: 10143560WCUT )
Cover slip 24 x 60 mm (Thermo Fisher Scientific, catalog number: 15747592 )
Compost (Proveen, catalog number: 14926 )
Cauliflower (Brassica oleracea var. botrytis, cultivar Clovis, Vilmorin)
Note: Plants were grown on compost in a controlled greenhouse (short day conditions 9 h light; temperature 22 °C; relative humidity 70%). After inoculation, they were placed back in the controlled greenhouse for 24 h inside the miniature greenhouse at 100% relative humidity. After 24 h, lid was removed. All the experiments used the second true leaf from four-week-old plants.
Xcc strain 8004::GUS-GFP (Xanthomonas campestris pv. campestris (Xcc); Cerutti et al., 2017)
Magnesium chloride (MgCl2) (Merck, catalog number: 814733 )
Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
Casamino acids (BD, BD Biosciences, catalog number: 223050 )
Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 105101 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: 105886 )
Silwet L-77 (CAAHMRO, catalog number: 115950H )
Calcofluor (Fluorescent Brightener 28) (Sigma-Aldrich, catalog number: F3543 )
MOKA medium (see Recipes)
Equipment
Miniature greenhouse purchased from a garden center (Nortene, catalog number: ME673714 )
Hollow punch (Harris, Uni-Core) (Electron Microscopy Sciences, catalog number: 69039-70 ) or razor blades
Confocal microscope (Leica Microsystems, model: Leica TCS SP2 or Leica TCS SP8 ) equipped with an argon laser (ray line at 488 nm), a Helium Neon laser at 633 nm and a diode laser at 405 nm
Water immersion lens 40x with a long working distance (N.A. = 0.8, working distance of 3,300 µm) (Leica Microsystems, catalog number: 50615 5) or water immersion lens 63x with a long working distance (N.A. = 0.9 and a working distance of 2,900 µm) (Leica Microsystems, catalog number: 506148 ) (Leica Mannheim, Germany)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Cerutti, A. and Jauneau, A. (2017). Capturing Z-stacked Confocal Images of Living Bacteria Entering Hydathode Pores of Cauliflower. Bio-protocol 7(20): e2451. DOI: 10.21769/BioProtoc.2451.
Cerutti, A., Jauneau, A., Auriac, M. C., Lauber, E., Martinez, Y., Chiarenza, S., Leonhardt, N., Berthomé, R. and Noël, L. D. (2017). Immunity at cauliflower hydathodes controls systemic infection by Xanthomonas campestris pv campestris. Plant Physiol 174(2): 700-716.
Download Citation in RIS Format
Category
Plant Science > Plant cell biology > Cell imaging
Microbiology > Microbe-host interactions > In vivo model
Cell Biology > Cell imaging > Confocal microscopy
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2,452 | https://bio-protocol.org/exchange/protocoldetail?id=2452&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Histochemical Preparations to Depict the Structure of Cauliflower Leaf Hydathodes
AC Aude Cerutti
MA Marie-Christine Auriac
LN Laurent D. Noël
Alain Jauneau
Published: Vol 7, Iss 20, Oct 20, 2017
DOI: 10.21769/BioProtoc.2452 Views: 7679
Reviewed by: Hiroyuki HiraiYunbing Ma
Original Research Article:
The authors used this protocol in Jun 2017
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Abstract
Hydathodes are plant organs present on leaf margins of a wide range of vascular plants and are the sites of guttation. Both anatomy and physiology of hydathodes are poorly documented. We have recently reported on the anatomy of cauliflower and Arabidopsis thaliana hydathodes and on their infection by the vascular pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) (Cerutti et al., 2017). Because hydathodes are natural infection routes for several pathogens, it is necessary to have a deep knowledge of their anatomy to further better interpret images of infected hydathodes. Here, we described different detailed protocols for gaining information on hydathode anatomy which are applicable to a wide range of plants (including monocots like barley and rice). Nomarsky and confocal microscopy were used to observe clarified thick samples. Optical microscopy in transmitted light and transmission electron microscopy were used to observed thin and ultrathin sections.
Keywords: Cauliflower Hydathode Xanthomonas
Background
In literature, different techniques were used to study hydathodes (Perrin, 1972; Chen and Chen, 2007; Wang et al., 2011; Singh, 2014). From light microscopy (on entire tissues or on section of resin-embedded samples) to scanning or transmission electron microscopy, a large panel of protocols and techniques was available. To our knowledge, these techniques were not used in combination and laser confocal microscopy was never used to depict hydathode structures. Moreover, we noticed variations from protocols to protocols. We presented here different techniques used in combination. They are well-adapted to cauliflower and Arabidopsis thaliana. They have been successfully applied to other plants like monocotyledons (barley and rice) and should be likely used to a larger variety of plant species. We encourage the users to apply these protocols to gain complementary information on the hydathode at different scales during infection.
Materials and Reagents
Microscope glass slides (Thermo Fisher Scientific, Superfrost, catalog number: 10143560WCUT )
Cover slip 24 x 60 mm (Thermo Fisher Scientific, catalog number: 15747592 )
Razor blades
Cauliflower (Brassica oleracea var. botrytis, cultivar Clovis)
Notes:
Cauliflower plants were grown in a controlled greenhouse.
All the experiments used the second true leaf from four-weeks-old plants.
Chloral hydrate (Sigma-Aldrich, catalog number: 23100 )
Glycerol (C3H8O3) (VWR, catalog number: 24387.326 )
Calcofluor (Fluorescent Brightener 28) (Sigma-Aldrich, catalog number: F3543 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Sodium cacodylate (Sigma-Aldrich, catalog number: C4945 )
Glutaraldehyde EM grade (Electron Microscopy Sciences, catalog number: 16214 )
Osmium tetroxide (OsO4) (Electron Microscopy Sciences, catalog number: 19150 )
Ethanol (C2H5OH) (Sigma-Aldrich, catalog number: 32221 )
Epon (Electron Microscopy Sciences, catalog number: 14120 )
Note: Composition of the resin: Embed-812 (45 g), DDSA (36 g), NMA (18 g) and BDMA (1.35 ml). Embedding kit in which accelerator DMP30 is replaced by BDMA (Electron Microscopy Sciences, catalog number: 11400 ).
Borax (Sigma-Aldrich, catalog number: B3545 )
Toluidine blue (RAL Diagnostics, catalog number: 361590 )
Methylene blue (Merck, catalog number: 159270 )
Basic fuchsin (Sigma-Aldrich, catalog number: 857343 )
Periodic acid (VWR, Prolabo, catalog number: 20.593.151 )
Acetic acid (CH3COOH) (CARLO ERBA Reagents, catalog number: 302016 )
Thiocarbohydrazide (Sigma-Aldrich, catalog number: 223220 )
Silver proteinate (Roques for histology) (Sigma-Aldrich, catalog number: 05495 )
Aceton (CH3COCH3) (Fisher Scientific, catalog number: 10395640 )
Clarification solution (see Recipes)
Fixation solution (see Recipes)
Sodium cacodylate buffer (see Recipes)
Borax solution with toluidine blue and methylene blue (see Recipes)
Basic fuchsin solution (see Recipes)
Periodic acid solution (see Recipes)
Silver proteinate solution (see Recipes)
Equipment
Hollow punch (Harris, Uni-Core) (Electron Microscopy Sciences, catalog number: 69039-70 )
Ultra-microtome (Leica Microsystems, Reichert-Jung, model: UltraCut E )
Optical microscope equipped for Nomarski (Leica Microsystems, model: DM IRB-E )
Confocal microscope (Leica Microsystems, model: Leica TCS SP2 ) equipped with a diode laser at 405 nm
Diaphragm pump vacuum or other vacuum sources
Stirrer hotplate (IKA)
Microwave apparatus (Leica Microsystems, model: EM AMW )
Flat bottom embedding capsule (Electron Microscopy Sciences, catalog number: 70021 )
Oven (Memmert, model: UFE500BO )
Histo diamond knife (Diatome Histo) for semi-thin sections (0.5 µm) to optical observations
Ultra diamond knife (Diatome Ultra 45) for ultra-thin sections (70-80 nm) to transmission electron microscopy
Hot plate (slides warmer) (C & A Scientific, Premiere, model: XH-2002 )
Optical microscope (ZEISS, model: Axioplan 2 )
CCD camera (ZEISS, model: AxioCam MRc )
Transmission electron microscope (Hitachi, model: HT7700 )
Gold grid (Electron Microscopy Sciences, catalog number: FCF200-Au )
Software
Pro Plus 4.0 Imaging software (Media Cybernetics, Silver Spring, MD, USA)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Cerutti, A., Auriac, M., Noël, L. D. and Jauneau, A. (2017). Histochemical Preparations to Depict the Structure of Cauliflower Leaf Hydathodes. Bio-protocol 7(20): e2452. DOI: 10.21769/BioProtoc.2452.
Cerutti, A., Jauneau, A., Auriac, M. C., Lauber, E., Martinez, Y., Chiarenza, S., Leonhardt, N., Berthomé, R. and Noël, L. D. (2017). Immunity at cauliflower hydathodes controls systemic infection by Xanthomonas campestris pv campestris. Plant Physiol 174(2): 700-716.
Download Citation in RIS Format
Category
Plant Science > Plant immunity > Host-microbe interactions
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Fixed-tissue imaging
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2,453 | https://bio-protocol.org/exchange/protocoldetail?id=2453&type=0 | # Bio-Protocol Content
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Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells
AG Adrian J. Giovannone
ER Elena Reales
PB Pallavi Bhattaram
AF Alberto Fraile-Ramos
TW Thomas Weimbs
Published: Vol 8, Iss 3, Feb 5, 2018
DOI: 10.21769/BioProtoc.2453 Views: 8097
Edited by: Gal Haimovich
Reviewed by: Alexandros Kokotos
Original Research Article:
The authors used this protocol in Oct 2017
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Oct 2017
Abstract
The uptake and trafficking of cell surface receptors can be monitored by a technique called ‘antibody-feeding’ which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.
Keywords: Antibody-feeding assay Syntaxin SNARE Internalization Apical-basolateral polarity Epithelial cells MDCK cells
Background
The SNARE protein Syntaxin 3 (Stx3) is known to establish apical-basolateral polarity in polarized epithelial cells (Low et al., 1996; Delgrossi et al., 1997; Weimbs et al., 1997; Low et al., 1998; Li et al., 2002; Low et al., 2006). Apical localization of Stx3 depends on a conserved targeting motif near the N-terminus of the protein (ter Beest et al., 2005; Sharma et al., 2006). A fraction of Stx3 is also known to localize to late endosomes/lysosomes in Madin-Darby Canine Kidney (MDCK) cells, which form tight-junctions, establish apical and basolateral polarity, and adopt a columnar morphology when grown to confluence. To determine the origin of this population of Stx3 and to investigate its endosomal trafficking in polarized MDCK cells, we designed an ‘antibody-feeding assay’ protocol. Using this assay and other experiments, we have shown that Stx3 is ubiquitinated at lysine residues in a basic juxtamembrane region and that ubiquitination facilitates the endocytosis of Stx3 from the basolateral membrane leading to trafficking to intraluminal vesicles of multivesicular bodies, and eventually secretion with exosomes (Giovannone et al., 2017). A non-ubiquitinatable mutant (Stx3-5R) exhibits decreased endosomal trafficking and exosomal secretion (Giovannone et al., 2017). Using an antibody-feeding assay, we can monitor where Stx3 is trafficked after being delivered to the apical or basolateral membrane. A cassette containing two Myc epitope tags and one hexa-histidine tag was added to the C-terminus of Stx3 by molecular cloning. These tags are exposed to the extracytoplasmic side of the plasma membrane when tagged Stx3 is present on the surface in transfected cells. We have previously shown that these C-terminal Myc2-His6 tags are accessible to the 9E10 anti-c-Myc monoclonal antibody and do not interfere with the known surface polarity of several syntaxins (Low et al., 2000; Kreitzer et al., 2003; Low et al., 2006; Sharma et al., 2006; Reales et al., 2011; Giovannone et al., 2017). Epitope-tagged Stx3 is stably expressed in MDCK cells using a tetracycline-controlled transcriptional activation system which allows using uninduced cells as a negative control. Cells are cultured on Transwell permeable membranes until fully polarized (Figure 1), incubated with anti-c-Myc antibody, and harvested for analysis by immunofluorescence microscopy at various time points. Cells were also incubated with anti-M6PR antibody (late endosomal marker). Lastly, cells were stained with DAPI and secondary antibodies against the 9E10 anti-c-Myc monoclonal antibody (for Stx3) and anti-M6PR antibody.
Figure 1. Schematic drawing of a Transwell polycarbonate membrane cell culture insert inside a well of a typical 12-well cell culture dish. Cells are cultured on top of the membrane and will form a tight monolayer that seals off the polycarbonate membrane thereby separating the apical media compartment from the basolateral media compartment.
Materials and Reagents
10 cm cell culture dishes (Corning, Falcon®, catalog number: 353003 )
10 ml serological pipette (VWR, catalog number: 89130-898 )
50 ml conical tubes (Corning, catalog number: 430290 )
12-well cell culture plates with 0.4 µm pore polycarbonate Transwell supports (Corning, catalog number: 3401 )
12-well cell culture plates (Corning, Costar®, catalog number: 3512 )
Razor blades (VWR, catalog number: 55411-050 )
Paper towels
Kimwipes (KCWW, Kimberly-Clark, catalog number: 34120 )
Parafilm (BEMIS, catalog number: PM996 )
Plastic pencil box with lid (VWR, catalog number: 500003-109 )
Manufacturer: Janitorial Supplies, catalog number: AVT34104 .
Microscope slides (any standard slides from any supplier will be fine)
Micro cover glasses, 18 x 18 mm, No. 1 thickness is 0.13 to 0.17 mm (VWR, catalog number: 48366-045 )
Madin-Darby Canine Kidney (MDCK) cells stably expressing C-terminally Myc2-His6-tagged Stx3
Note: Madin-Darby Canine Kidney (MDCK) cells stably expressing C-terminally Myc2-His6-tagged Stx3 using a doxycycline-inducible expression system have been described in detail in Sharma et al., 2006. The parental cell line, expressing the TET transactivator is required to produce doxycycline-inducible, stably transfected cells for one’s gene of interest and have been generated in the laboratory of the senior author (TW). Cells may be requested from the authors. Original MDCK cells and several subclones are also available from ATCC (ATCC, catalog number: CCL-34 and others) but these cells may differ in some characteristics from those used here.
Sterile 1x Dulbecco’s phosphate buffered saline (DPBS) without calcium or magnesium (Mediatech, catalog number: 21-031-CV )
Sterile 0.25% trypsin/EDTA (Mediatech, catalog number: 22-053-CI )
4% buffered formalin solution (Sigma-Aldrich, catalog number: HT5012 )
Prolong Gold anti-fade reagent plus DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36934 )
Sterile Minimum Essential Medium without glutamine (Mediatech, catalog number: 15-010-CV )
Sterile L-glutamine 100x (Mediatech, catalog number: 25-005-CI )
Penicillin/Streptomycin 100x (Mediatech, catalog number: 30-002-CI )
Fetal bovine serum (FBS) (Omega Scientific, catalog number: FB-11 )
Doxycycline monohydrate (tetracycline analog) (Sigma-Aldrich, catalog number: D1822 )
HEPES (Fisher Scientific, catalog number: BP310 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
Anti-c-Myc monoclonal antibody, clone 9E10
Note: Hybridoma cells from The Developmental Studies Hybridoma Bank (http://dshb.biology.uiowa.edu). Prepare mouse ascites using a commercial vendor.
Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A5666 )
Note: This product has been discontinued.
L-glycine (Fisher Scientific, catalog number: BP381-5 )
Normal Donkey serum (LAMPIRE Biological Labs, catalog number: 7332100 )
Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
Mannose-6-Phosphate Receptor antibody (kind gift from William Brown, Cornell University)
Fish skin gelatin (Sigma-Aldrich, catalog number: G7765 )
Donkey anti-mouse DyLight 488 (Jackson ImmunoResearch Laboratories, catalog number: 715-485-150 )
Note: This product has been discontinued.
Donkey anti-rabbit DyLight 594 (Jackson ImmunoResearch Laboratories, catalog number: 711-515-152 )
Note: This product has been discontinued.
Complete media (see Recipes)
Serum-free media (see Recipes)
1 mg/ml doxycycline stock solution (see Recipes)
MEM ETC (see Recipes)
MEM ETC containing anti-c-Myc antibody (see Recipes)
Quench solution (see Recipes)
Block and Permeabilization buffer (see Recipes)
Primary antibody solution (see Recipes)
Washing solution (see Recipes)
Secondary antibody solution (see Recipes)
Equipment
BSL-2 hood (Nuaire, class II)
Tissue culture incubator at 37 °C and 5% CO2 (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150 )
Orbital shaker (Bellco)
Hemocytometer (Hausser Scientific, catalog number: 3200 )
Surgical scissors 140 mm (Dunrite Instruments, catalog number: 140940 )
Dissecting forceps, fine tip, non-serrated
Olympus Fluoview FV1000S (Olympus, model: Fluoview FV1000 ) Spectral Laser Scanning Confocal microscope using an Olympus UPLFLN 60x oil-immersion objective
Cold room (4 °C)
Software
Adobe Photoshop (Adobe Systems Inc.)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Giovannone, A. J., Reales, E., Bhattaram, P., Fraile-Ramos, A. and Weimbs, T. (2018). Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells. Bio-protocol 8(3): e2453. DOI: 10.21769/BioProtoc.2453.
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Category
Cell Biology > Cell imaging > Fixed-cell imaging
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2,455 | https://bio-protocol.org/exchange/protocoldetail?id=2455&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle
JM Joseph D. McMillan
ME Michael A. Eisenback
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2455 Views: 17194
Edited by: Antoine de Morree
Reviewed by: Sesha Lakshmi Arathi Paluri
Original Research Article:
The authors used this protocol in Dec 2010
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The authors used this protocol in:
Dec 2010
Abstract
Skeletal muscle is the most abundant tissue in the human body and regulates a variety of functions including locomotion and whole-body metabolism. Skeletal muscle has a plethora of mitochondria, the organelles that are essential for aerobic generation of ATP which provides the chemical energy to fuel vital functions such as contraction. The number of mitochondria in skeletal muscle and their function decline with normal aging and in various neuromuscular diseases and in catabolic conditions such as cancer, starvation, denervation, and immobilization. Moreover, compromised mitochondrial function is also associated with metabolic disorders including type 2 diabetes mellitus. It is now clear that maintaining mitochondrial content and function in skeletal muscle is vital for sustained health throughout the lifespan. While a number of staining methods are available to study mitochondria, transmission electron microscopy (TEM) is still the most important method to study mitochondrial structure and health in skeletal muscle. It provides critical information about mitochondrial content, cristae density, organization, formation of autophagosomes, and any other abnormalities commonly observed in various disease conditions. In this article, we describe a detailed protocol for sample preparation and analysis of mouse skeletal muscle mitochondria by TEM.
Keywords: Transmission electron microscopy Skeletal muscle Mitochondria Autophagy Myopathy Atrophy Oxidative metabolism
Background
Skeletal muscle is a highly plastic tissue that undergoes morphological and metabolic adaptations in response to a number of extracellular cues. A number of perturbations including resistance or endurance exercise stimulates mitochondrial biogenesis leading to increased metabolic capacity and resistance to fatigue (Li et al., 2008; Sandri, 2008). By contrast, during aging, inactivity, and in many catabolic disease states, skeletal muscle mitochondrial number and function decline, leading to increased fatigability and insulin resistance (Sandri, 2008). An accumulation of dysfunctional mitochondria may also result in progressive reactive oxygen species-induced damage, producing a further impairment of oxidative capacity in skeletal muscle (Bonnard et al., 2008).
Mitochondria exist as a reticular membrane network that is located in different subcellular compartments in skeletal muscle. The subsarcolemmal (SS) mitochondria, account for 10-15% of the mitochondrial volume and lie directly beneath the sarcolemmal membrane, whereas the intermyofibrillar (IMF) mitochondria are located in close contact with the myofibril (Takahashi and Hood, 1996). Mitochondria are double membrane structures containing an intermembrane space between the outer and inner membranes as well as the inner matrix compartment, where most of the metabolic processes take place. The inner membrane is highly folded, forming so-called cristae, to accommodate its large surface area. The five complexes that make up the respiratory chain where oxidative phosphorylation takes place are embedded within the inner mitochondrial membrane. In this process, a proton gradient across the inner membrane is coupled to ATP synthesis at complex V (Peterson et al., 2012). In addition to producing ATP for cross-bridge cycling between actin and myosin, mitochondria are a source of free radicals which regulate skeletal muscle physiology (Peterson et al., 2012).
Transmission electron microscopy (TEM) is a powerful technique for ultrastructural studies (Watson, 1958). TEM has been very useful in studying mitochondrial structure in skeletal muscle in both physiological and pathological conditions (Picard et al., 2013). For example, TEM can provide information about mitochondrial content, organization, cristae structure, and vacuolization as observed in some neuromuscular disorders such as Amyotrophic lateral sclerosis (Picard et al., 2013). In many muscle wasting conditions, mitochondrial content is reduced through autophagy, also known as mitophagy. In this regard, TEM has been found to be an important approach to study autophagosome formation (Sandri, 2008). We have developed an efficient protocol that can be easily adapted in any laboratory to study the ultrastructure of mouse mitochondria in skeletal muscle by TEM (Paul et al., 2010; Hindi et al., 2014 and 2018). In the following sections, we provide a step-wise protocol for sample preparation and analysis of SS and IMF mitochondria in skeletal muscle. A similar protocol can be used for studying other organelles in skeletal muscle by TEM as well.
Materials and Reagents
Glass specimen vials (Electron Microscopy Sciences, catalog number: 72630-05 )
Razor blades, Double Edge Coated, Washed Version (Electron Microscopy Sciences, Personna, catalog number: 72000-WA )
Transfer pipette (Fisher Scientific, catalog number: 13-711-9BM )
Nitrile gloves
Glass strips, ultramicrotomy grade, 6.4 x 25 x 400 mm (Electron microscopy sciences, catalog number: 71012 )
Glass slides (Fisher Scientific, catalog number: 12-550-15 )
Syringes 1 ml (BD, catalog number: 329652 )
Syringes 30 ml (BD, catalog number: 302833 )
0.22 μm syringe filter (Merck, catalog number: SLGV033RS )
Conical centrifuge tube, 50 ml (VWR, catalog number: 21008-169 )
Conical centrifuge tube, 15 ml (VWR, catalog number: 21008-089 )
Non-sterile urine specimen container (Electron Microscopy Sciences, catalog number: 64231-10 )
Filter paper, Qualitative Grade 1 Circles (GE Healthcare, Whatman, catalog number: 1001-090 )
Glass stirring rod (United Scientific Supplies, catalog number: GSR012 )
Wood applicators (Electron microscopy Sciences, catalog number: 72300 )
Flat, silicone embedding mold (Electron Microscopy Sciences, catalog number: 70900 )
Glass knife boat, 6.4 mm (Electron Microscopy Sciences, catalog number: 71007 )
Glass knife box (Electron Microscopy Sciences, catalog number: 71010 )
N95 respirator, with valve (VWR, catalog number: 89201-510 )
Metal loop, perfect loop (Electron Microscopy Sciences, catalog number: 70944 )
Grids, tabbed, copper, 200 mesh (Ted Pella, catalog number: 3HGC200 )
Grid storage box, tabbed (Ted Pella, catalog number: 161 )
Petri dish, glass, 100 x 20 mm (Corning, catalog number: 70165-102 )
Glutaraldehyde, EM grade, 8% (Polysciences, catalog number: 00216-30 )
Sodium phosphate monobasic monohydrate, NaH2PO4·H2O (Sigma-Aldrich, catalog number: S9638 )
Sodium phosphate dibasic anhydrous, Na2HPO4 (Sigma-Aldrich, catalog number: S9763 )
Osmium tetroxide, 10 x 1 g (Electron Microscopy Sciences, catalog number: 19110 )
Ethanol, 200 Proof (Decon Labs, catalog number: 2701 )
EMbed-812 kit, includes: EMbed-812, DDSA, NMA, and DMP-30 (Electron Microscopy Sciences, catalog number: 14120 )
Sodium borate (MP Biomedicals, catalog number: 0219030980 )
Toluidine blue O (Amresco, catalog number: 0672-25G )
Uranyl acetate dihydrate powder (depleted) (Electron Microscopy Sciences, catalog number: 22400 )
NaOH pellets (Amresco, catalog number: 0583-500G )
Lead Nitrate, Pb(NO3)2 (Electron Microscopy Sciences, catalog number: 17900 )
Sodium citrate, Na3(C6H5O7)·2H2O (Electron Microscopy Sciences, catalog number: 21140 )
Propylene oxide, EM grade (Electron Microscopy Sciences, catalog number: 20401 )
Dental wax (Electron Microscopy Sciences, catalog number: 72660 )
3% glutaraldehyde (see Recipe 1)
0.1 M sodium phosphate buffer pH 7.4 (see Recipe 2)
1% osmium tetroxide (see Recipe 3)
Ethanol dilutions (see Recipe 4)
Embedding media (see Recipe 5)
1% toluidine blue stain (see Recipe 6)
4% uranyl acetate stock (aq) (see Recipe 7)
1 N-NaOH (see Recipe 8)
Reynold’s Lead Citrate (see Recipe 9)
Precautions/Hazards: As with any chemicals and reagents handled in the lab, users should be aware of how to use and manipulate them safely. Please refer to each chemical’s Material Safety Data Sheet (MSDS) for detailed information about precautions and hazards. Electron microscopy uses quite a few hazardous chemicals, such as: glutaraldehyde, osmium tetroxide, propylene oxide, uranyl acetate, lead citrate, and others. Please handle these chemicals using the proper personal protective equipment (PPE), ventilation conditions, and dispose of these chemicals in accordance with your institution’s Department of Environmental Health and Safety.
Equipment
Amber, wide-mouth glass bottle, 125 ml (VWR, catalog number: 10861-846 )
Clear, media bottle, 1 L (Corning, PYREX®, catalog number: 1399-1L )
Graduated cylinder, 1,000 ml (VWR, catalog number: 65000-012 )
Graduated cylinder, 25 ml (VWR, catalog number: 65000-002 )
Magnetic stirring bar (VWR, catalog number: 58948-025 )
General-Purpose Liquid-In-Glass Thermometer (VWR, catalog number: 89095-626 )
Negative-action, curved self-closing tweezers (Electron Microscopy Sciences, catalog number: 72864-D )
Eyelash manipulator (Electron Microscopy Sciences, catalog number: 71182 )
1,000 ml Glass Griffin beaker (VWR, catalog number: 10754-960 )
50 ml Glass Griffin beakers (VWR, catalog number: 10754-946 )
Glass funnel, 100 mm (VWR, catalog number: 10546-048 )
50 ml volumetric flask (VWR, catalog number: 10123-996 )
100 ml volumetric flask, amber (VWR, catalog number: 10124-022 )
-20 °C Freezer (VWR, catalog number: 97014-903 )
4 °C Refrigerator (VWR, catalog number: 14236-525 )
Clinical rotator, variable speed tube rotator (Cole-Parmer, Stuart, catalog number: SB3 )
Culture tube holder for clinical rotator, variable speed tube rotator, 12 mm offering a rolling action for tubes (Cole-Parmer, Stuart, catalog number: SB3/3 )
pH meter, SymPhony B10P (VWR, catalog number: 89231-662 )
pH probe, refillable, glass (VWR, catalog number: 89231-580 )
Precision Balance, AV212C (Ohaus, out of production)
Precision Balance (OHAUS, catalog number: 30122632 )
Hotplate stirrer (Fisher Scientific, catalog number: SP88857200P )
Vacuum oven (Electron Microscopy Sciences, catalog number: 63235-10 )
Ultramicrotome (Reichert-Jung, model: Ultracut E , out of production, eBay or other second-hand markets)
New ultramicrotome (Leica Microsystems, model: Leica EM UC7 )
Glass knifemaker (LKB, model: LKB Type 7801B , out of production, eBay or other second-hand markets)
New glass knifemaker, Leica EM KMR3 (Leica Microsystems,, catalog number: Leica EM KMR3 )
Light microscope, Olympus CX31 (Olympus, catalog number: CX31 )
Diamond knife, Diatome wet ultra 45°, 3.5 mm (Electron Microscopy Sciences, model: Diatome Ultra )
Phillips CM10 transmission electron microscope retrofitted with a new digital camera (Phillips, catalog number: CM10 )
High Definition CCD Camera for TEM retrofitted to Phillips CM10 scope (Advanced Microscopy Techniques, catalog number: BioSprint )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
McMillan, J. D. and Eisenback, M. A. (2018). Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle. Bio-protocol 8(10): e2455. DOI: 10.21769/BioProtoc.2455.
Paul, P. K., Gupta, S. K., Bhatnagar, S., Panguluri, S. K., Darnay, B. G., Choi, Y. and Kumar, A. (2010). Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice. J Cell Biol 191(7): 1395-1411.
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Category
Cell Biology > Cell structure > Cell organelle
Developmental Biology > Morphogenesis > Cell structure
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2,456 | https://bio-protocol.org/exchange/protocoldetail?id=2456&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Context-driven Salt Seeking Test (Rats)
SC Stephen E. Chang
KS Kyle S. Smith
Published: Vol 8, Iss 7, Apr 5, 2018
DOI: 10.21769/BioProtoc.2456 Views: 5386
Reviewed by: Beatriz CastroXi Feng
Original Research Article:
The authors used this protocol in Jun 2017
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The authors used this protocol in:
Jun 2017
Abstract
Changes in reward seeking behavior often occur through incremental learning based on the difference between what is expected and what actually happens. Behavioral flexibility of this sort requires experience with rewards as better or worse than expected. However, there are some instances in which behavior can change through non-incremental learning, which requires no further experience with an outcome. Such an example of non-incremental learning is the salt appetite phenomenon. In this case, animals such as rats will immediately seek out a highly-concentrated salt solution that was previously undesired when they are put in a novel state of sodium deprivation. Importantly, this adaptive salt-seeking behavior occurs despite the fact that the rats never tasted salt in the depleted state, and therefore never tasted it as a highly desirable reward.
The following protocol is a method to investigate the neural circuitry mediating adaptive salt seeking using a conditioned place preference (CPP) procedure. The procedure is designed to provide an opportunity to discover possible dissociations between the neural circuitry mediating salt seeking and salt consumption to replenish the bodily deficit after sodium depletion. Additionally, this procedure is amenable to incorporating a number of neurobiological techniques for studying the brain basis of this behavior.
Keywords: Salt appetite Conditioned place preference
Background
The salt appetite phenomenon was first discovered by Richter (1936), who found that rats began to immediately consume a 1% salt solution in greater quantities compared to water in a 2-bottle choice test following adrenalectomy and consequently bodily sodium depletion. More recently, Robinson and Berridge (2013) have shown that this immediate increase in salt-seeking behavior occurs to discrete salt-paired cues following sodium depletion in the absence of salt before it has been tasted as a desirable reward. In addition, adaptive salt-seeking has also been observed with contextual cues following sodium depletion (Stouffer and White, 2005).
In the brain, there is clear evidence that the central nucleus of the amygdala (Galaverna et al., 1993; Seeley et al., 1993; Tandon et al., 2012; Hu et al., 2015), lateral hypothalamus (Wolf and Quartermain, 1967; Tandon et al., 2012), and the nucleus accumbens (Roitman et al., 2002; Voorhies and Bernstein, 2006; Loriaux et al., 2011; Tandon et al., 2012) are important for the consumption of salt following sodium depletion in order for animals to replenish the deficit. However, there has been surprisingly little work done on the neural circuitry mediating cue-driven salt seeking following sodium depletion. In other words, it is mostly unclear how the brain enables animals to seek out salt in a novel deprivation state. In a recent study, we showed that the ventral pallidum (VP) plays an important role in this phenomenon (Chang et al., 2017). The VP has previously been shown to track the changes in value of salt-paired cues before and after sodium depletion (Tindell et al., 2009; Robinson and Berridge, 2013). However, it was previously unknown whether the VP is necessary for mediating salt appetite in terms of cue-driven salt seeking or salt consumption. Using a novel CPP procedure, described here, we showed that optogenetic inhibition of the VP impairs context-driven salt seeking but not the consumption of salt itself following sodium depletion (Chang et al., 2017).
The protocol we have used to demonstrate this effect allows for not only optogenetics to be used but also other techniques to manipulate the brain (e.g., DREADDs, intracranial injections, lesions) or to record brain activity (e.g., electrophysiology, calcium imaging). Further study of context-driven salt seeking with this procedure may help elucidate the neural bases of disorders of aberrant motivation that may lead to reduced reward seeking, as in depression, or non-homeostatic reward seeking (e.g., overeating leading to obesity). In addition, this procedure could be easily extended to investigate the neural bases of other nutrient deficit-induced changes in behavior such as calcium appetite (Leshem et al., 1999; Schulkin, 2000).
Materials and Reagents
Electrical tape (TemflexTM 1700, 3M, catalog number: 1700-1X66FT )
Disposable weighing boat
Rat (Long-Evans, 250-300 g; Male; 7 weeks old; Charles River Laboratories)
Sodium-free food (TestDiet; Order Info: 1816123 (5ANR), TD 90228 1/2)
Sugar (pure cane granulated, Domino®)
Salt (iodized, Morton Salt, Inc.)
Sugar-free Kool-Aid powder (orange and grape, Kool-Aid, Kraft Foods, Inc.)
Furosemide (Salix®, Merck)
Equipment
Preference test chamber (29.5 x 12.5 x 21 in., custom designed; Dartmouth Apparatus Shop)
16 oz. screw-top bottles (Ancare, catalog number: MST16 )
Screw-top bend ball pt. tubes (5 in. long with 1 in. bend, Ancare, catalog number: PCST51BTD )
Digital video camera (Sony)
2 portable luminaires (UL, catalog number: E196460 )
2 red light bulbs (Sunlite, catalog number: SL24/R )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Chang, S. E. and Smith, K. S. (2018). Context-driven Salt Seeking Test (Rats). Bio-protocol 8(7): e2456. DOI: 10.21769/BioProtoc.2456.
Chang, S. E., Smedley, E. B., Stansfield, K. J., Stott, J. J. and Smith, K. S. (2017). Optogenetic inhibition of ventral pallidum neurons impairs context-driven salt seeking. J Neurosci 37(23): 5670-5680.
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Category
Neuroscience > Behavioral neuroscience > Learning and memory
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2,458 | https://bio-protocol.org/exchange/protocoldetail?id=2458&type=0 | # Bio-Protocol Content
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Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq
MR Mauricio A. Reynoso*
GP Germain C. Pauluzzi*
SC Sean Cabanlit
JV Joel Velasco
JB Jérémie Bazin
RD Roger Deal
SB Siobhan Brady
NS Neelima Sinha
JB Julia Bailey-Serres
Kaisa Kajala
*Contributed equally to this work
Published: Vol 8, Iss 7, Apr 5, 2018
DOI: 10.21769/BioProtoc.2458 Views: 14405
Edited by: Renate Weizbauer
Reviewed by: Juliane K IshidaHiroyuki Hirai
Original Research Article:
The authors used this protocol in Feb 2018
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Abstract
Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would not capture non-polyadenylated pre-mRNA, we also present our method to remove ribosomal RNA from the total nuclear RNA in preparation for nuclear RNA-Seq.
Keywords: Gene expression Nuclear purification INTACT rRNA degradation RNA extraction RNA-Seq
Background
Isolating specific cell types for gene expression experiments reduces the noise and increases the precision of the experiment and the number of differently expressed genes found. Various methods for cell type-specific studies are widely used, each one with their strengths and weaknesses (reviewed in Bailey-Serres, 2013). Isolating specific regulatory compartments, such as nuclei (from organelles, ribosomes, cytosol, etc.) provides further precision in dissecting the molecular events in regulation of gene expression. Here we describe a method that allows isolating nuclei of specific cell types from frozen tissue, suited for experiments where nuclear gene expression is to be studied (e.g., RNA-Seq of nuclear RNA, ATAC-Seq, ChIP-Seq, etc.). Furthermore, we describe the processing of RNA that leads to material suited to be the input for an RNA-Seq experiment. The protocols described here were used with rice root tissue (Oryza sativa cv. Nipponbare) (Reynoso et al., 2018), but they are based on previous protocols developed for Arabidopsis (Deal and Henikoff, 2010 and 2011) and tomato (Ron et al., 2014).
The first part of this protocol, the INTACT method (for Isolation of Nuclei Tagged in specific Cell Types) allows in vivo affinity labeling and subsequent purification of nuclei from a cell type of interest. This is achieved through cell type-specific expression of a tripartite nuclear tagging fusion protein (NTF) consisting of a nuclear envelope targeting domain, GFP, and the biotin ligase recognition peptide (BLRP). Co-expression of NTF along with the E. coli biotin ligase gene codon optimized for rice, BirA, in the cell type of interest results in the production of fluorescently labeled, biotinylated nuclei specifically in that cell type. These labeled nuclei can then be affinity purified from a crude tissue homogenate using streptavidin-coated magnetic beads, thus allowing access to RNA and chromatin from the cell type of interest.
The second part of the method describes the processing of nuclear RNA to produce a sample that is suitable to be the input for RNA-Seq library preparation. Typically for eukaryotic samples, ribosomal RNA (rRNA) and organellar RNA is removed from RNA samples by polyA-pulldown methods. However, nuclear mRNA contains pre-mRNA in many stages of processing, most not polyadenylated, while many of the polyadenylated mRNAs are rapidly exported. Hence, to profile the transcripts at different stages of processing, polyA-isolation methods are not suited. Inspired by previous work (Morlan et al., 2012; Gregory Smaldone, personal communication), we developed a method to remove ribosomal RNA (rRNA) from rice samples, specifically using the INTACT-purified nuclei as our input. The steps of this method include isolation of total RNA from INTACT-purified nuclei, removal of residual genomic DNA, annealing of DNA probes to rRNA, degradation of rRNA with RNase specific to RNA:DNA hybrids, and degradation of the DNA probes. At the end of the protocol, the RNA sample is ready for RNA-Seq library construction.
Part I: INTACT purification of nuclei
Materials and Reagents
Pipette tips (P20, P200 and P1000, nuclease-free, e.g., USA Scientific, catalog numbers: 1123-1710 , 1120-8780 , 1126-7510 )
(Optional) 13 ml Falcon tube
30 μm cell strainer (Sysmex, CellTrics, catalog number: 04-004-2326 )
15 ml Falcon tubes (nuclease-free, e.g., VWR, catalog number: 89039-666 )
1.5 ml Eppendorf tubes (nuclease-free, e.g., Denville Scientific, catalog number: C2170 )
Pasteur pipettes (nuclease-free, e.g., Phenix Research Products, catalog number: PP-137038C )
Slide
Coverslip
PCR tube strips (nuclease-free, e.g., USA Scientific, catalog number: 1402-2708 )
0.22 μm syringe filters (e.g., Merck, catalog number: SLGP033RB )
50 ml Falcon tubes (nuclease-free, e.g., VWR, catalog number: 89039-658 )
Aluminum foil
Transgenic plant tissue with biotin-tagged nuclei
INTACT binary vectors (Ron et al., 2014; Reynoso et al., 2018)
Note: They will be available at https://gateway.psb.ugent.be/search. These plasmids allow inserting a T-DNA with the promoter of your choice into the plant species of your choice.
Liquid nitrogen
M-280 Streptavidin Dynabeads (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11205D )
NaOH
MOPS (Sigma-Aldrich, catalog number: M1254-25G )
Sodium chloride (NaCl) (e.g., Fisher Scientific, catalog number: S271 )
Potassium chloride (KCl) (e.g., Fisher Scientific, catalog number: BP366-1 )
Ethylenediaminetetraacetic acid (EDTA) (e.g., Fisher Scientific, catalog number: S311 )
EGTA (Sigma-Aldrich, catalog number: E3889-25G )
Spermine (Sigma-Aldrich, catalog number: S1141 )
Spermidine (Sigma-Aldrich, catalog number: S2626 )
Protease Inhibitor Cocktail (Sigma-Aldrich, catalog number: P9599 ) or cOmplete mini protease inhibitor tablets EDTA-free (Roche Diagnostics, catalog number: 11836170001 )
Triton X-100 (Sigma-Aldrich, catalog number: 648466 )
Propidium Iodide (PI) stain (Sigma-Aldrich, catalog number: P4170 )
10 N NaOH (see Recipes)
0.5 M MOPS, pH 7 (see Recipes)
1 M NaCl (see Recipes)
2 M KCl (see Recipes)
0.5 M EDTA (see Recipes)
0.5 M EGTA (see Recipes)
0.2 M spermine (see Recipes)
0.2 M spermidine (see Recipes)
10% Triton X-100 (see Recipes)
Nuclei purification buffer base (see Recipes)
NPB and NPBt buffers (see Recipes)
Propidium Iodide (PI) stains (see Recipes)
Equipment
Pipettes (P20, P200, P1000; e.g., Eppendorf, catalog numbers: 3123000039 , 3123000055 , 3123000063 )
Ceramic mortars and pestles (wipe clean with RNaseZap [Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 ] or other RNase removal product)
Centrifuge for 15 ml Falcon tubes (e.g., Eppendorf, model: 5810 R )
DynamagTM-15 Magnet (Thermo Fisher Scientific, model: DynamagTM-15, catalog number: 12301D )
Note: Can be replaced with a homemade version. For example, a strong rare earth (neodymium) magnet (e.g., 1 x 10 cm bar) can be taped inside a 50 ml Falcon tube and padding can be added to keep 15 ml Falcon tubes in place next to the magnet (see Figure 1).
Figure 1. Homemade magnet for two 15 ml Falcon tubes. Two neodymium magnets are placed inside 50 ml Falcon tubes held in a tube rack. The attraction between the two magnets holds them in place. To prop the 15 ml tubes to correct position against the magnet, paper towel is taped inside the 50 ml tube.
DynamagTM-2 Magnet (Thermo Fisher Scientific, model: DynamagTM-2, catalog number: 12321D ), or a homemade version of this
Hemocytometer (SCK Films, In-Cyto, catalog number: DHC-N01-2 )
BD AdamsTM NutatorTM Single-Speed Orbital Mixer (e.g., Fisher Scientific, catalog number: 14-062)
Manufacturer: BD, catalog number: 421105/DEL .
Fridge and/or cold room at 4 °C
Fluorescent microscope with filters to visualize either DAPI or PI stain (Edmund Optics, catalog numbers: 86-371 , 67-008 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Reynoso, M. A., Pauluzzi, G. C., Cabanlit, S., Velasco, J., Bazin, J., Deal, R., Brady, S., Sinha, N., Bailey-Serres, J. and Kajala, K. (2018). Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq. Bio-protocol 8(7): e2458. DOI: 10.21769/BioProtoc.2458.
Reynoso, M.A, Pauluzzi, G., Kajala, K., Cabanlit, S., Velasco, J., Bazin, J., Deal, R.B., Sinha, N.R., Brady, S.M., Bailey-Serres J. (2018). Nuclear transcriptomes at high resolution using retooled INTACT. Plant Physiol 176(1): 270-281.
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Category
Plant Science > Plant molecular biology > RNA
Plant Science > Plant molecular biology > RNA
Molecular Biology > RNA > RNA sequencing
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246 | https://bio-protocol.org/exchange/protocoldetail?id=246&type=0 | # Bio-Protocol Content
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Construction and Screening of a Transposon Insertion Library of Yersinia enterocolitica (YeO3-R1)
MP Maria Pajunen
EP Elise Pinta
Mikael Skurnik
Published: Vol 2, Iss 15, Aug 5, 2012
DOI: 10.21769/BioProtoc.246 Views: 14283
Original Research Article:
The authors used this protocol in Jan 2012
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Abstract
The Mu-transposon system is one of the best characterized transposition systems. Under minimal in vitro set-up, Mu transposition requires only a simple reaction buffer, MuA transposase protein, mini-Mu transposon DNA (donor) and target DNA. The reaction proceeds via initial assembly of the transposition complex that directs transposon integration into target DNA with high efficiency and relatively low target site selectivity. These characteristics make the Mu in vitro transposition technology ideal for the generation of comprehensive mutant DNA libraries usable in a variety of molecular biology applications. This technology has successfully been used for DNA sequencing, functional analyses of plasmid DNA and virus genomes, protein engineering for structure/function and protein-protein interaction studies and generation of gene targeting constructions. When electroporated, the in vitro–assembled Mu transposition complexes can also be used for efficient gene delivery in bacteria, yeasts and mammalian cells. Using this protocol we have identified several mutants where Cat-Mu insertion has interrupted genes involved in lipopolysaccharide (LPS) biosynthesis (Pinta et al., 2012).
Keywords: Yersinia enterocolitica Mu transposase Transposon insertion library Lipopolysaccharide Bacteriophage
Materials and Reagents
Yersinia enterocolitica strain YeO3-R1 (al-Hendy et al., 1992)
Cat-Mu transposon (Haapa et al., 1999) or equivalent like Entranceposon Cam-R3 (Thermo Fisher Scientific, Finnzymes, catalog number: F-778 )
MuA transposase 1,100 ng/μl and MuA storage buffer (Thermo Fisher Scientific, Finnzymes, catalog number: F-750C )
99.5% glycerol BDH (catalog number: 24388.320 )
10% Triton X-100 (w/v) (F. Hoffmann-La Roche, catalog number: 11332481001 )
Ultrapure 0.5 M EDTA (pH 8.0) (Life Technologies, Invitrogen™, catalog number: 15575 )
DTT (Sigma-Aldrich, catalog number: D0632 )
Centricon YM-100 (100-kDa cutt off) (EMD Millipore, catalog number: UFC210024PL )
Polyethylene glycol (PEG) 6000 (Merck KGaA, catalog number: 8.07491 )
NuSieve 3:1 Agarose (25 g) (Lonza, catalog number: 50091 )
Albumin from bovine serum, BSA (Sigma-Aldrich, catalog number: A7906 )
Heparin (Sigma-Aldrich, catalog number: H3393 )
Ficoll PM 400 (Sigma-Aldrich, catalog number: F4375 )
Bacto-agar, Bacto-tryptone, and Bacto-yeast extract (BD DifcoTM)
Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
Tryptic Soya Broth (Oxoid Limited, catalog number: CM0129 )
Enterocoliticin produced by Y. enterocolitica serotype O: 7, 8 strain 29930 (Strauch et al., 2001)
MuA storage buffer
Sodium acetate (NaAc)
DNA ladder (New England Biolabs)
Phosphate-buffered saline (PBS) (pH 7.4)
TGD buffer (see Recipes)
5x complex buffer (see Recipes)
1 M DTT (see Recipes)
20% PEG6000 (w/v) – 2.5 M NaCl (5 ml) (see Recipes)
SOB medium (1 L) without magnesium (see Recipes)
2 M Mg++ stock (see Recipes)
2 M glucose (100 ml) (see Recipes)
SOC medium (100 ml) (see Recipes)
Luria broth (LB) medium (1 L) (see Recipes)
LB agar (LA) plates (1 L) (see Recipes)
Chloramphenicol (Clm) solution (10 ml) (see Recipes)
Tryptic soya broth (see Recipes)
Phosphate-buffered saline (PBS) (pH 7.4) (1 L) (see Recipes)
TAE buffer (see Recipes)
Equipment
Centrifuges (Sorvall, Heraeus Holding)
Water bath (Grant)
Incubator (Termaks)
Shaker (New Brunswick Scientific)
Biofotometer (Eppendorf)
Agarose gel electrophoresis (Bio-Rad Laboratories)
Gel documentation equipment (Bio-Rad Laboratories)
Electroporator (Bio-Rad Laboratories, Genepulser II)
90 mm Petri dishes (Thermo Fisher Scientific, Sterilin®, catalog number: 101RT )
0.1-cm electroporation cuvettes (Bio-Rad Laboratories)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pajunen, M., Pinta, E. and Skurnik, M. (2012). Construction and Screening of a Transposon Insertion Library of Yersinia enterocolitica (YeO3-R1). Bio-protocol 2(15): e246. DOI: 10.21769/BioProtoc.246.
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Category
Microbiology > Microbial genetics > Mutagenesis
Molecular Biology > DNA > Mutagenesis
Systems Biology > Genomics > Transposons
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2,467 | https://bio-protocol.org/exchange/protocoldetail?id=2467&type=0 | # Bio-Protocol Content
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Ex vivo Whole-cell Recordings in Adult Drosophila Brain
Alexa J. Roemmich
Soleil S. Schutte
Diane K. O’Dowd
Published: Vol 8, Iss 14, Jul 20, 2018
DOI: 10.21769/BioProtoc.2467 Views: 6529
Reviewed by: Steven Boeynaems
Original Research Article:
The authors used this protocol in Jan 2006
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Abstract
Cost-effective and efficient, the fruit fly (Drosophila melanogaster) has been used to make many key discoveries in the field of neuroscience and to model a number of neurological disorders. Great strides in understanding have been made using sophisticated molecular genetic tools and behavioral assays. Functional analysis of neural activity was initially limited to the neuromuscular junction (NMJ) and in the central nervous system (CNS) of embryos and larvae. Elucidating the cellular mechanisms underlying neurological processes and disorders in the mature nervous system have been more challenging due to difficulty in recording from neurons in adult brains. To this aim we developed an ex vivo preparation in which a whole brain is isolated from the head capsule of an adult fly and placed in a recording chamber. With this preparation, whole cell recording of identified neurons in the adult brain can be combined with genetic, pharmacological and environmental manipulations to explore cellular mechanisms of neuronal function and dysfunction. It also serves as an important platform for evaluating the mechanism of action of new therapies identified through behavioral assays for treating neurological diseases. Here we present our protocol for ex vivo preparations and whole-cell recordings in the adult Drosophila brain.
Keywords: Adult brain dissection Drosophila Electrophysiology Antennae lobe High temperature recording
Background
The fruit fly (Drosophila melanogaster) has been used to make key discoveries in a variety of fundamental areas in neuroscience including learning and memory (Bolduc et al., 2008; Cervantes-Sandoval et al., 2016), synapse formation and regulation (Genç et al., 2017), and circadian rhythms (Allada et al., 1998; Guo et al., 2016). Mutants identified through both forward and reverse genetic screens have also provided useful models of human neurological disorders including Fragile X syndrome, Parkinson’s Disease, Huntington Disease and epilepsy disorders (Pallos et al., 2008; Parker et al., 2011; Liu et al., 2012; Sears and Broadie, 2017). Much of what we have learned in this research comes from electrophysiological recordings and calcium imaging at the neuromuscular junction (NMJ), from neurons in dissociated primary culture, or from the central nervous system of embryos and larvae. Though these methods have been instrumental in our understanding, to elucidate the underlying cellular mechanisms of neurological processes in adult animals, it is important to have electrophysiological access to individual neurons of the adult brain.
Recordings from neurons in the adult CNS were made possible by development of two complementary systems in the mid-2000s. One involves exposing and desheathing a small area of brain making it possible to obtain intracellular recordings from neurons in a live, behaving adult fly (Wilson et al., 2004; Hige et al., 2015; Nagel and Wilson, 2016). This preparation is best suited to recording from populations of neurons on the dorsal surface of the brain. The second preparation involves removing the whole brain from the adult head capsule and placing it in a recording chamber (Gu and O’Dowd, 2006 and 2007). This provides access to neurons in the entire brain and allows for easy environmental manipulations. Although the process is invasive and may cause damage to the brain, intact neurons and functional circuits can be persevered and maintained for up to one hour after skillful dissection. Labs have used whole brain dissection and whole-cell recordings to characterize the electrical properties of circadian neurons (Sheeba et al., 2008b), uncover the electrical cellular mechanisms responsible for sleep and arousal (Sheeba et al., 2008a), discover a new light-sensing pathway in the brain (Ni et al., 2017), determine the mechanism of action for a common pesticide (Qiao et al., 2014), find a memory suppressor miRNA that regulates an autism susceptibility gene (Guven-Ozkan et al., 2016), and describe synaptic dysfunction in a model of Parkinson’s Disease (Sun et al., 2016).
Our lab uses this protocol extensively to study the cellular mechanisms of genetic epilepsy associated with mutations in SCN1A, a gene that encodes NaV1.1 sodium channels that are highly expressed in inhibitory, GABAergic neurons in the human brain. Using homologous recombination, and more recently CRISPR/Cas9 mediated gene editing, we have introduced specific SCN1A missense mutations into the same location in the Drosophila sodium channel gene, para. We have shown that all of the mutations causing febrile seizure phenotypes in humans that we have examined (K1270T, S1231R, R1648H/C), also result in heat-induced seizure phenotypes in the adult fly (Sun et al., 2012; Schutte et al., 2014 and 2016). To evaluate how specific mutations alter sodium currents and neuronal activity, we perform electrophysiological analyses of sodium currents in knock-in flies carrying SCN1A mutations, focused primarily on GABAergic, local neurons (LNs) in the antennal lobe. Whole-cell recordings from the cell bodies of LNs can be used to evaluate sodium currents and firing properties in mutant compared to wild-type neurons. The ability to rapidly exchange extracellular recording solutions in the ex vivo preparation allows fast and reversible elevation of the temperature to assess constitutive and temperature-dependent changes in sodium currents and firing properties in knock-in mutant compared to wild-type neurons. Fast perfusion also facilitates evaluation of the acute effects of potential anti-convulsant drugs on sodium currents and firing properties. Here we present our updated protocol for ex vivo whole-cell recordings in adult Drosophila brains, including fly dissection and preparation, data acquisition, and analysis.
Materials and Reagents
Materials
Ex vivo preparation
35 mm Petri dish
1cc Plastic syringes
27 G ½ needles
Electrophysiology
Plastic tubing for perfusion system
0.5 mm diameter platinum wire
0.07 mm diameter nylon fiber
Recording chamber and platform (such as Harvard Apparatus, models: RC-27 and PH-6D )
Borosilicate glass micropipettes (100 μl) (such as VWR, catalog number: 53432-921 )
Reagents
Ex vivo preparation
Sodium chloride (NaCl) (1 M) (Sigma-Aldrich, catalog number: S5886 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C4901 )
Magnesium chloride solution (MgCl2) (1 M) (Sigma-Aldrich, catalog number: M1028 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
L-cysteine (Sigma-Aldrich, catalog number: C7352 )
Papain suspension (Worthington Biochemical, catalog number: LS003126 )
Sodium hydroxide (NaOH) (10 N) (Fisher Scientific, catalog number: S25550 )
Electrophysiology
Colbalt (II) chloride (CoCl2) (Sigma-Aldrich, catalog number: 60818 )
Tetraethylammonium chloride (TEA) (Sigma-Aldrich, catalog number: T2265 )
4-aminopyridine (4-AP) (Sigma-Aldrich, catalog number: 275875 )
(+)-Tubocurarine chloride (curarine) (Tocris Bioscience, catalog number: 2820 )
Picrotoxin (Sigma-Aldrich, catalog number: P1675 )
Potassium gluconate (Kgluconate) (Sigma-Aldrich, catalog number: P1847 )
EGTA (Sigma-Aldrich, catalog number: E4378 )
Adenosine 5’-triphosphate disodium salt hydrate (Na2ATP) (Sigma-Aldrich, catalog number: A2383 )
Potassium hydroxide (KOH) (8 N) (Sigma-Aldrich, catalog number: P4494 )
Cesium hydroxide (CsOH) (50 wt. % in H2O) (Sigma-Aldrich, catalog number: 232068 )
D-gluconic acid solution (49-53 wt. % in H2O) (Sigma-Aldrich, catalog number: G1951 )
Equipment
Ex vivo preparation
AA Forceps
Fine-tip tweezers
Osmometer (such as Wescor, model: 5600 )
Dissecting stereomicroscope (such as Nikon Instruments, model: SMZ800N )
Gooseneck light source (such as Edmund Optics, model: Fiber-Lite® Illuminator System, catalog number: 35-277 )
Electrophysiology
Pipette puller (such as NARISHIGE, model: PC-100 )
Upright microscope (such as OLYMPUS, model: BX51WI )
Amplifier (such as Molecular Devices, model: Axopatch 200B )
Digitizer (such as Molecular Devices, model: Digidata 1500B )
Micromanipulator (such as Sutter Instrument, model: MP-225 )
Air table and Faraday cage (such as Sutter Instrument, model: AT-3036 )
Peristaltic pump system (such as Cole-Parmer, catalog number: EW-77910-20 )
(Additional) In-Line Solution Heater (Harvard Apparatus, model: SHM-828 )
(Additional) Temperature Controller (Harvard Apparatus, model: CL-100 )
Software
pClamp software suite (minimum version 9.0)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Roemmich, A. J., Schutte, S. S. and O'Dowd, D. K. (2018). Ex vivo Whole-cell Recordings in Adult Drosophila Brain. Bio-protocol 8(14): e2467. DOI: 10.21769/BioProtoc.2467.
Gu, H. and O'Dowd, D. K. (2006). Cholinergic synaptic transmission in adult Drosophila Kenyon cells in situ. J Neurosci 26(1): 265-272.
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Category
Neuroscience > Nervous system disorders > Cellular mechanisms
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247 | https://bio-protocol.org/exchange/protocoldetail?id=247&type=0 | # Bio-Protocol Content
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Senescence Associated β-galactosidase Staining
Michael Eccles
CL Caiyun Grace Li
Published: Vol 2, Iss 16, Aug 20, 2012
DOI: 10.21769/BioProtoc.247 Views: 70343
Original Research Article:
The authors used this protocol in Dec 2011
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Abstract
Detection of senescent cells using a cytochemical assay was first described in 1995 (Dimri et al., 1995). The identification of senescent cells is based on an increased level of lysosomal β-galactosidase activity (Kurz et al., 2000). Cells under normal growth condition produce acid lysosomal β- galactosidase, which is localized in the lysosome. The enzymatic activity can be detected at the optimal pH 4.0, using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal) (Miller, 1972). In comparison, upon senescence, the lysosomal mass is increased, leading to production of a higher level of β-galactosidase, termed senescence-associated β-galactosidase (SA-β-gal) (Kurz et al., 2000). The abundant senescence-associated enzyme is detectable over background despite the less favorable pH conditions (pH 6.0) (Dimri et al., 1995). The SA-β gal positive cells stain blue-green, which can be scored under bright-field microscopy. In this assay it is best to avoid over-confluency of the cells, or cells that have undergone too many passages, as these conditions can cause false positive results.
Keywords: Senescence Beta-galactosidase Colormetric
Materials and Reagents
Paraformaldehyde (PFA) (Sigma-Aldrich)
5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal) (Sigma-Aldrich)
Potassium ferrocyanide (Sigma-Aldrich, catalog number: B4252 )
Potassium ferricyanide (Sigma-Aldrich, catalog number: P9387 )
Phosphate buffered saline (PBS)
Sodium hydroxide
Dimethylformamide
Sodium chloride
Magnesium chloride
Dibasic sodium phosphate
Citric acid
Sodium phosphate
4% paraformaldehyde (PFA) (see Recipes)
Senescence associated β-galactosidase (SA-β-gal) staining solution (see Recipes)
Equipment
Inverted microscope [e.g. Olympus 1 x 71 inverted microscope (Olympus)]
24-well plate
p1000 pipette
37 °C incubator
Hot plate
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Eccles, M. and Li, C. G. (2012). Senescence Associated β-galactosidase Staining. Bio-protocol 2(16): e247. DOI: 10.21769/BioProtoc.247.
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Category
Cancer Biology > Cell death > Cell biology assays
Cell Biology > Cell viability > Cell death
Cell Biology > Cell staining > Whole cell
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2,470 | https://bio-protocol.org/exchange/protocoldetail?id=2470&type=0 | # Bio-Protocol Content
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Peer-reviewed
Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method
TC Tetsuo Cai
TT Taisuke Tomita
Published: Vol 8, Iss 17, Sep 5, 2018
DOI: 10.21769/BioProtoc.2470 Views: 5505
Original Research Article:
The authors used this protocol in Dec 2017
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Dec 2017
Abstract
Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine residues facing the hydrophilic environment, even within the transmembrane domain. Cysteine crosslinking experiments provide us with information about the distance between two cysteine residues. The combination of these methods enables us to obtain information about the structural changes of the target protein. Here, we describe the detailed protocol for structural analysis using SCAM.
Keywords: Cysteine Structural analysis Substitution Water accessibility Biotinylation Membrane protein Crosslinking
Background
Structural analyses provide the critical information about the function of a target protein. X-ray crystallography and nuclear magnetic resonance have been utilized as high-resolution protein structural analysis methods in the biology field. However, these methods require a purified protein extracted from membrane at very high concentration for the structural analysis of membrane proteins. Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to analyze the water accessibility and the spatial distance of particular cysteine residues substituted in the target protein. Using methanethiosulfonate (MTS) reagents that specifically react with the cysteine residues facing the hydrophilic environment, we can annotate the topology and structure of the target protein. As the labeling reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) is impermeable to the plasma membrane (Seal et al., 1998), in intact cells, only extracellular, but not intracellular, cysteine residues are biotinylated (Figure 1A). In contrast, both extracellular and intracellular cysteine residues are exposed to the hydrophilic environment in microsomes and are biotinylated by MTSEA-biotin (Figure 1B). Moreover, the accessibility of cysteine can be analyzed by competition experiments using membrane-impermeable MTS derivatives (Figure 2A). By combining the results of these analyses, we are able to obtain structural information of the entire intrinsic protein in the membrane (Akabas et al., 1992; Loo and Clarke, 1995; Frillingos et al., 1998; Karlin and Akabas, 1998; Foucaud et al., 2001; Kaback et al., 2001; Bogdanov et al., 2005; Sato et al., 2006 and 2008; Takagi et al., 2010; Watanabe et al., 2010; Takagi-Niidome et al., 2015; Tominaga et al., 2016; Cai et al., 2017; Tomita, 2017). The accessibility of cysteine can be analyzed in more detail by competition experiments using membrane-impermeable MTS derivatives; the negatively charged 2-sulfonatoethyl methanethiosulfonate (MTSES), the positively charged 2-(trimethylammonium)-ethyl methanethiosulfonate (MTSET), and the sterically bulkiest 2-(Triethylammonium)-Ethyl Methanethiosulfonate Bromide (MTS-TEAE) (Figure 2A). Moreover, cross-linking experiments using microsome fractions provide information regarding the spatial distance between two separated cysteines in different polypeptides (Figure 3A) (Loo and Clarke, 1996; 2000; 2001; Klco et al., 2003). In this experiment, all endogenous cysteines of the target protein should be mutated by serine or alanine (cys-less mutant), and then one (single cys-mutation) or two (double cys-mutation) target residues would be substituted to the cysteine to analyze the accessibility of particular residue(s) by MTS reagents. However, sometimes these substitutions affect the structure and function of the target protein. Thus, it is important to analyze the biological function of the target protein to ensure whether the mutations do not affect the protein conformation.
Materials and Reagents
QSP 0.1-10 μl PIPETTE TIP (Thermo Fisher Scientific, Quality Scientific Plastics, catalog number: 102-Q )
1-200 μl New Pipette Tip Yellow (Thermo Fisher Scientific, Quality Scientific Plastics, catalog number: TW110-Q )
101-1,000 μl Pipette Tip Blue (Thermo Fisher Scientific, Quality Scientific Plastics, catalog number: 111-Q )
1,000-5,000 μl Pipette Tip Natural (Thermo Fisher Scientific, Quality Scientific Plastics, catalog number: 090-Q )
15 ml centrifuge tube (IWAKI, catalog number: 2325-015 )
12-well flat bottom microplate (IWAKI, catalog number: 3815-012 )
150 mm tissue culture dish (IWAKI, catalog number: 3030-150 )
1 ml syringe (TERUMO, catalog number: SS-01T )
27 G needle (TERUMO, catalog number: NN-2719S )
MEF cells
Immunostar® Reagents (Immunostar; Wako Pure Chemical Industries, catalog number: 291-55203 )
SuperSignalTM West Femto Maximum Sensitivity Substrate (Supersignal; Thermo Fisher Scientific, catalog number: 34096 )
Streptavidin SepharoseTM High Performance (SA beads; GE Healthcare, catalog number: 17511301 )
Disodium hydrogenphosphate 12-Water (Na2HPO4•12H2O; Wako Pure Chemical Industries, catalog number: 196-02835 )
Sodium dihydrogenphosphate Dihydrate (NaH2PO4•2H2O; Wako Pure Chemical Industries, catalog number: 192-02815 )
Potassium chloride (KCl; Wako Pure Chemical Industries, catalog number: 163-03545 )
Sodium chloride (NaCl; NACALAI TESQUE, catalog number: 31320-34 )
Potassium dihydrogen phosphate (KH2PO4; Wako Pure Chemical Industries, catalog number: 169-04245 )
Copper(II) sulfate (CuSO4; Wako Pure Chemical Industries, catalog number: 034-04445 )
Sodium Dodecyl Sulfate (SDS; NACALAI TESQUE, catalog number: 31606-04 )
1,10-Phenanthroline monohydrate (Phenanthroline; Wako Pure Chemical Industries, catalog number: 169-00862 )
N-Ethylmaleimide (NEM; NACALAI TESQUE, catalog number: 15512-11 )
cOmpleteTM Protease inhibitor cocktail (Roche Diagnostics, catalog number: 11836145001 )
2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES; Dojindo Molecular Technologies, catalog number: GB10 )
O,O'-Bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA; Dojindo Molecular Technologies, catalog number: G002 )
Sucrose (Wako Pure Chemical Industries, catalog number: 196-00015 )
Tris (hydroxymethyl) aminomethane (Tris; C4H11NO3; STAR CHEMICAL, catalog number: RSP-THA500G )
Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
Brilliant Green (Wako Pure Chemical Industries, catalog number: 021-02352 )
Coomassie Brilliant Blue G-250 (CBB G-250; NACALAI TESQUE, catalog number: 09409-42 )
DMEM, high glucose, no glutamate, no methionine, no cysteine (DMEM (cys-); Thermo Fisher Scientific, catalog number: 21013-024 )
Dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries, catalog number: 045-28335 )
2-mercaptoethanol (Wako Pure Chemical Industries, catalog number: 139-07525 )
N-Biotinylaminoethyl Methanethiosulfonate (MTSEA-biotin; Toronto Research Chemicals, catalog number: B394750 )
Sodium 2-Sulfonatoethyl Methanethiosulfonate (MTSES; Toronto Research Chemicals, catalog number: S672000 )
2-(Trimethylammonium)-Ethyl Methanethiosulfonate Bromide (MTSET; Toronto Research Chemicals, catalog number: T795900 )
2-(Triethylammonium)-Ethyl Methanethiosulfonate Bromide (MTS-TEAE; Toronto Research Chemicals, catalog number: T775800 )
1,2-Ethanediyl Bismethanethiosulfonate (M2M; Toronto Research Chemicals, catalog number: E890350 )
1,3-Propanediyl Bismethanethiosulfonate (M3M; Toronto Research Chemicals, catalog number: P760350 )
1,4-Butanediyl Bismethanethiosulfonate (M4M; Toronto Research Chemicals, catalog number: B690150 )
1,6-Hexanediyl Bismethanethiosulfonate (M6M; Toronto Research Chemicals, catalog number: H294250 )
3,6-Dioxaoctane-1,8-diyl Bismethanethiosulfonate (M8M; Toronto Research Chemicals, catalog number: D486150 )
Undecane-1,11-diyl-bismethanethiosulfonate (M11M; Toronto Research Chemicals, catalog number: U787800 )
3,6,9,12-Tetraoxatetradecane-1,14-diyl-bis-methanethiosulfonate (M14M; Toronto Research Chemicals, catalog number: T306250 )
3,6,9,12,15-Pentaoxaheptadecane-1,17-diyl Bis-methanethiosulfonate (M17M; Toronto Research Chemicals, catalog number: P273750 )
1x Phosphate Buffered Saline (1x PBS; see Recipes)
1x Dulbecco's Phosphate Buffered Saline (1x DPBS; see Recipes)
1x Sample buffer (sample buffer; see Recipes)
1x homogenize buffer (see Recipes)
Equipment
0.5-10 μl Pipettor (NICHIRYO, model: Nichipet EXII, catalog number: 00-NPX2-10 )
2-20 μl Pipettor (NICHIRYO, model: Nichipet EXII, catalog number: 00-NPX2-20 )
20-200 μl Pipettor (NICHIRYO, model: Nichipet EXII, catalog number: 00-NPX2-200 )
100-1,000 μl Pipettor (NICHIRYO, model: Nichipet EXII, catalog number: 00-NPX2-1000 )
1,000-5,000 μl Pipettor (NICHIRYO, model: Nichipet EXII, catalog number: 00-NPX2-5000 )
Forma Series II Water Jacket CO2 Incubator (Thermo Fisher Scientific, catalog number: 3110 )
Polytron homogenizer (Hitachi, model: HG30 )
500-Watt ultrasonic processor (Sonics & Materials, model: VCX 500 )
Small size culture rotator (TAITEC, model: RT-50 )
High speed centrifuge (Koki Holdings, himac, model: CF15RN )
High speed refrigerated micro centrifuge (TOMY, model: MX-307 )
Ultracentrifuge (Beckman Coulter, model: Optima L-90K )
Ultracentrifuge fixed angle rotor (Beckman Coulter, model: Type 50.4 Ti , catalog number: 347299)
Aluminum block bath (TAITEC, model: DTU-1CN )
ImageQuant LAS 4000 (GE Healthcare)
Centrifuge tube (Beckman Coulter, catalog number: 355645 )
Spatula
Software
ImageQuant TL 7.0 (GE Healthcare)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Cai, T. and Tomita, T. (2018). Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method. Bio-protocol 8(17): e2470. DOI: 10.21769/BioProtoc.2470.
Cai, T., Yonaga, M. and Tomita, T. (2017). Activation of γ-secretase trimming activity by topological changes of transmembrane domain 1 of presenilin 1. J Neurosci 37(50): 12272-12280.
Download Citation in RIS Format
Category
Biochemistry > Protein > Structure
Biochemistry > Protein > Activity
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2,471 | https://bio-protocol.org/exchange/protocoldetail?id=2471&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Image-Based Analysis of Mitochondrial Area and Counting from Adult Mouse Dopaminergic Neurites
NN Nadee Nissanka
CM Carlos T Moraes
MP Mlena Pinto
Published: Vol 8, Iss 16, Aug 20, 2018
DOI: 10.21769/BioProtoc.2471 Views: 7890
Original Research Article:
The authors used this protocol in Jan 2018
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Original research article
The authors used this protocol in:
Jan 2018
Abstract
Mitochondria form dynamic cytoplasmic networks which undergo morphological changes in order to adapt to cellular stresses and signals. These changes can include alterations in size and number within a given cell. Analysis of the whole network can be a useful metric to assess overall mitochondrial health, particularly in neurons, which are highly sensitive to mitochondrial dysfunction. Here we describe a method which combines immunofluorescence and computerized image analysis to measure mitochondrial morphology (quantification of number, density, and area) in dopaminergic neurites of mice expressing mitochondrially-targeted eYFP.
Keywords: Neurite mitochondria Mitochondrial morphology Mitochondrial size Neurite length FIJI
Background
Mitochondria are double-membraned organelles present in essentially all the cells of every complex organism. Their main function is to supply the majority of cellular energy as ATP, but they also play roles in apoptosis, buffering intracellular Ca2+, reactive oxygen species production, and regulation of membrane potential (Neupert and Herrmann, 2007; Hamanaka and Chandel, 2010; Shutt and McBride, 2013).
These organelles, often depicted as single “bean-like” structures, are in reality components of a dynamic cytoplasmic network. They can undergo major morphological changes regulated by dynamic processes of membrane fusion and fission, a process believed to be involved in the elimination of dysfunctional organelles through a process called mitophagy. The mitochondrial network can also increase as a response to high cellular energy demand (Sheng, 2017; Devine and Kittler, 2018). The morphology of the mitochondrial network can be altered in response to different stressors, and there is a wide range of possible morphologies which are cell-type, or even cell-compartment dependent (Picard et al., 2013). The localization of mitochondria in the dendrites and axons of neurons, in particular, play a crucial role as they provide, in these specific cells, the energy necessary for synaptic transmission (Chang and Reynolds, 2006; Misgeld and Schwarz, 2017).
Mitochondria localization and dynamics in one particular group of cells, the dopaminergic neurons in the substantia nigra, have been extensively studied during the last decades. These neurons’ projections reach the striatum and their loss causes a depletion of striatal dopamine which is the cause of the classical motor symptoms of Parkinson’s disease (PD) (Dauer and Przedborski, 2003; Braak et al., 2004).
The involvement of mitochondrial dysfunctions in these neurons has been investigated since the 80’s (Kopin and Markey, 1988), but the study of mitochondrial dynamics attracted particular interest especially since the discovery that genes mutated in monogenic forms of Parkinson’s disease (in particular Parkin and PINK1) have an essential role in mitochondrial fission/fusion, mitochondrial transport and mitophagy (Koh and Chung, 2010; Narendra and Youle, 2011).
We recently investigated the consequences of the loss of Parkin in a mouse model of PD in which degeneration of dopaminergic neurons was caused by mtDNA depletion and mitochondrial dysfunction (Pinto et al., 2018). In this context, we also found that lack of Parkin affected mitochondrial morphology in dopaminergic axons.
A challenge in studying the mitochondria morphology in vivo is the clear visualization of the organelles and in discerning the ones present in the axons. To study mitochondrial morphology in dopaminergic neurons, we used immunofluorescence microscopy on mice specifically expressing eYFP in the mitochondria of dopaminergic neurons, as well as computerized image analysis software to study mitochondrial number, density and size, as a measure of mitochondrial health (Pinto et al., 2018).
Materials and Reagents
500 ml Vacuum Filter (Corning, catalog number: 431097 )
Single edge razor blades (VWR, catalog number: 55411-050 )
24-well Tissue Culture Plate (VWR, catalog number: 10062-896 )
Microscope Cover Glasses (VWR, catalog number: 16004-098 )
Micro Slides, Superfrost Plus (VWR, catalog number: 48311-703 )
Syringes (Insulin Syringes) (BD, catalog number: 328468 )
Mouse strains: males mito-eYFP DAT-tTA were analyzed at 4 months of age
mito-eYFP (C57BL/6-Tg(tetO-COX8A/eYFP)1Ksn/J) (THE JACKSON LABORATORIES, catalog number: 006618 )
DAT-tTA (B6;129S-Slc6a3tm4.1(tTA)Xz/J) (THE JACKSON LABORATORIES, catalog number: 027178 )
Super glue (Henkel, Loctite, catalog number: 1399967 )
Ketamine (Ketamine HCl, Hospira, catalog number: 02051-05 )
Xylazine (AnaSed, NDC 59599-110-20)
Reagent Alcohol (Sigma-Aldrich, catalog number: 793213 )
Paraformaldehyde (Sigma-Aldrich, catalog number: 441244 )
10x PBS (Merck, Calbiochem, catalog number: 6505-4L )
Hard Set Fluorescent Mounting Medium (Vector Laboratories, catalog number: H-1400 )
Sodium azide (Sigma-Aldrich, catalog number: S8032 )
1x PBS (see Recipes)
4% Paraformaldehyde (see Recipes)
0.02% (w/v) Sodium azide (see Recipes)
Equipment
Perfusion machine (Gilson, model: MINIPULS® 3 )
23 G Surshield Safety Winged Infusion Set (Terumo Medical, catalog number: SV*S23BL )
Blunt scissors (Thermo Fisher Scientific, catalog number: 78702 )
Sharp scissors (Fisher Scientific, catalog number: 13-808-2 )
Forceps (Fisher Scientific, catalog number: 13-812-39 )
Metal Spatula (Fisher Scientific, catalog number: 14-374 )
Acrylic Brain Matrix for Adult Mouse, Coronal Slices, 1 mm spacing (World Precision Instruments, catalog number: RBMA-200C )
Vibratome (Leica Biosystems, model: Leica VT1000 S )
Confocal Microscope (ZEISS, Laser Scanning Microscope LSM 710 Observer Z1)
Software
Zen Software Package (ZEN 2010B SP1, Version 6,0,0,485, Configuration 5.00.01)
FIJI (FIJI Is Just ImageJ) (Version 2.0.0–rc–67/1.52d)
Notes:
It is a distribution package of ImageJ with plugins for image analysis
It is available for free download here (https://imagej.net/Fiji/Downloads)
Microsoft® Excel
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Nissanka, N., Moraes, C. T. and Pinto, M. (2018). Image-Based Analysis of Mitochondrial Area and Counting from Adult Mouse Dopaminergic Neurites. Bio-protocol 8(16): e2471. DOI: 10.21769/BioProtoc.2471.
Pinto, M., Nissanka, N. and Moraes, C. T. (2018). Lack of Parkin anticipates the phenotype and affects mitochondrial morphology and mtDNA levels in a mouse model of Parkinson's disease. J Neurosci 38(4): 1042-1053.
Download Citation in RIS Format
Category
Neuroscience > Cellular mechanisms > Mitochondria
Cell Biology > Cell imaging > Fixed-tissue imaging
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2,472 | https://bio-protocol.org/exchange/protocoldetail?id=2472&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Quantitative Heterokaryon Assay to Measure the Nucleocytoplasmic Shuttling of Proteins
FM François McNicoll
Michaela Müller-McNicoll
Published: Vol 8, Iss 17, Sep 5, 2018
DOI: 10.21769/BioProtoc.2472 Views: 8313
Original Research Article:
The authors used this protocol in Jul 2017
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Abstract
Many proteins appear exclusively nuclear at steady-state but in fact shuttle continuously back and forth between the nucleus and the cytoplasm. For example, nuclear RNA-binding proteins (RBPs) often accompany mRNAs to the cytoplasm, where they can regulate subcellular localization, translation and/or decay of their cargos before shuttling back to the nucleus. Nucleocytoplasmic shuttling must be tightly regulated, as mislocalization of several RBPs with prion-like domains such as FUS and TDP-43 causes the cytoplasmic accumulation of solid pathological aggregates that have been implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Traditionally, interspecies heterokaryon assays have been used to determine whether a nuclear protein of interest shuttles; those assays are based on the fusion between donor and recipient cells from two different species (e.g., mouse and human), which can be distinguished based on different chromatin staining patterns, and detecting the appearance of the protein in the recipient nucleus. However, identification of heterokaryons requires experience and is prone to error, which makes it difficult to obtain high-quality data for quantitative studies. Moreover, transient overexpression of fluorescently tagged RBPs in donor cells often leads to their aberrant subcellular localization. Here, we present a quantitative assay where stable donor cell lines expressing near-physiological levels of eGFP-tagged RBPs are fused to recipient cells expressing the membrane marker CAAX-mCherry, allowing to readily identify and image a large number of high-confidence heterokaryons. Our assay can be used to measure the shuttling activity of any nuclear protein of interest in different cell types, under different cellular conditions or between mutant proteins.
Keywords: RNA-binding protein Nucleocytoplasmic shuttling Heterokaryon assay Quantification of shuttling capacities Nuclear proteins
Background
To understand the various functions of a protein, it is important to find out where it localizes within cells. Standard microscopic and biochemical methods only reveal the presence of a protein when its steady-state concentration is above the detection threshold. They do not rule out the possibility that it plays additional, important roles where it localizes only transiently (Gama-Carvalho and Carmo-Fonseca, 2001). For example, many RBPs perform functions in different cellular compartments where they accompany their bound mRNAs (often going undetected) and connect multiple steps in eukaryotic gene expression (Müller-McNicoll and Neugebauer, 2013). SR proteins (SRSF1 to SRSF12) are a family of RBPs that regulate transcription, pre-mRNA splicing, 3’end processing and mRNP packaging in the nucleus and appear exclusively nuclear at steady state (Howard and Sanford, 2015; Jeong, 2017). However, most family members shuttle continuously (but to different extents) between the nucleus and the cytoplasm, performing additional functions in mRNA export and translation (Caceres et al., 1998; Sapra et al., 2009; Maslon et al., 2014; Müller-McNicoll et al., 2016; Botti et al., 2017). Changes in RBP shuttling have been described in viral infections, early development, cellular differentiation and neurodegenerative diseases such as ALS and FTD, where pathological accumulation of prion-like RBPs such as FUS and TDP-43 in the cytoplasm forms solid neurotoxic aggregates (Ederle and Dormann, 2017; Liu et al., 2017). Thus, it is very important to know whether an RBP normally shuttles between the nucleus and the cytoplasm and if so, under which circumstances and how it is controlled.
With the tools currently available, it has been difficult to study the cytoplasmic functions of nuclear RBPs and to compare their shuttling abilities. An ingenious method was developed almost thirty years ago–the interspecies heterokaryon assay–in which donor and recipient cells from two different species (e.g., mouse and human) are fused and a protein present only in the donor nuclei gradually appears in the recipient nuclei if it shuttles (Borer et al., 1989). However, this assay provides only qualitative information. The fusion events are identified based on phase-contrast images and donor and recipient nuclei are identified based on distinct chromatin features, which makes the assay laborious, subjective and produces only small numbers of high-confidence heterokaryons. Moreover, fluorescently tagged RBPs are often expressed from transiently transfected plasmids, which results in very different RBP levels in different cells, ranging from barely detectable to non-physiologically high expression that may lead to partially aberrant cytoplasmic or subnuclear localization of RBPs [(Maharana et al., 2018) our unpublished observations]. Altogether, these limitations preclude any comparative analyses.
Here, we present a detailed experimental protocol to perform quantitative shuttling assays in cultured mammalian cells (Figure 1). Our assay is an improvement of the classical heterokaryon assay, with two novelties and a standardized imaging pipeline to perform quantitative measurements. The first novelty is the use of recipient cell lines expressing a fluorescently tagged membrane marker (CAAX-mCherry), which greatly facilitates the identification of heterokaryons containing both a donor and a recipient nucleus and thus allows the rapid and easy identification of a large number of high-confidence heterokaryons. The second novelty is the use of stable clonal donor cell lines, where a fluorescently (eGFP) tagged RBP of interest is expressed from a bacterial artificial chromosome (BAC), which has been integrated into the genome (Botti et al., 2017; Poser et al., 2008). Subsequent clonal selection of cells ensures equal and near-physiological levels of tagged RBPs in every donor cell to facilitate image acquisition, analysis and comparisons.
Our assay has been successfully applied to compare the shuttling activities of different SR proteins in the same cell line, between different cell lines and between differentiation states. Moreover, it allowed us to study the requirements for the shuttling of individual SR proteins using mutated proteins and knockdowns of nuclear export factors (Botti et al., 2017). We have used various cell lines as either donor or recipient cells: these comprise mouse (P19 and NIH3T3) and human (HeLa) cells. Although other cell lines remain to be tested, we are confident that any adherent cell line in which fluorescent RBPs can be expressed at physiological levels is suitable for our assay. This should include primary cells obtained from transgenic animals expressing a fluorescently tagged RBP of interest. We have successfully used P19 cells differentiated into neural cells as donors in our assays (Botti et al., 2017), and it should be possible to study and quantify shuttling of RBPs in other cellular models of differentiation, for example in mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or to compare shuttling of RBPs in distinct cellular differentiation fates (Hammarskjold and Rekosh, 2017). Moreover, our assay should allow to quantify changes in shuttling during viral infections and cellular stress, or to assess the impact of disease mutations in RBPs. In principle, our assay could even be adapted to visualize shuttling of long-noncoding RNAs (lncRNAs), for example through the insertion of binding sites for fluorescent MS2 binding protein (MS2-BP) or by inserting an aptamer sequence that binds a fluorescent dye (Ouellet, 2016).
Materials and Reagents
10 μl filter tips long (SARSTEDT, catalog number: 70.1116.210 )
20 μl filter tips (SARSTEDT, catalog number: 70.760.213 )
300 μl filter tips (SARSTEDT, catalog number: 70.765.210 )
1,000 μl filter tips (SARSTEDT, catalog number: 70.762.211 )
10-cm cell culture dishes (VWR, Thermo Fisher Scientific, catalog number: 734-2043 )
Cloning discs, size 5 mm (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z374458-100EA )
Disposable Glass Pasteur Pipettes 150 mm (VWR, catalog number: 612-1701 )
Serological pipettes, 2 ml (VWR, Corning, catalog number: 734-1690 )
Serological pipettes, 5 ml (VWR, Corning, catalog number: 734-1737 )
15-ml Centrifuge tubes (Corning, catalog number: 430791 )
2-ml microcentrifuge tubes (SARSTEDT, catalog number: 72.691 )
1,000 µl pipette tips ART® 1000E Barrier Tips (Thermo Fisher Scientific, catalog number: 2079E )
12-well plates for cell culture (VWR, Thermo Fisher Scientific, catalog number: 734-2156 )
Precision coverslips, 18 mm, borosilicate glass 0.17 ± 0.005 mm (Carl Roth, catalog number: LH23.1 )
Microscope slides (VWR, catalog number: 631-1550 )
Bacterial artificial chromosome (BAC) containing the gene encoding an eGFP-tagged RBP of interest (Botti et al., 2017 and Poser et al., 2008; see Notes 1-3)
Plasmid for expression of fluorescent plasma membrane marker of a different color (e.g., CAAX-mCherry, plasmid TH0477, Stewart et al., 2011; Botti et al., 2017; see Note 4)
DMEM, high glucose, GlutaMAXTM Supplement, pyruvate (Thermo Fisher Scientific, catalog number: 31966047 )
Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 10270106 )
Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, catalog number: 15140122 )
Puromycin 10 mg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: A1113803 )
Geneticin® Selective Antibiotic (G418 Sulfate) (50 mg/ml) (Thermo Fisher Scientific, catalog number: 10131035 )
Trypsin 0.05% EDTA (Thermo Fisher Scientific, catalog number: 25300054 )
Dulbecco's Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8537 )
Gelatin solution bioreagent 2% in H2O (Sigma-Aldrich, catalog number: G1393 )
Cycloheximide solution 100 mg/ml in DMSO (Sigma-Aldrich, catalog number: C4859 )
Polyethylene Glycol (PEG) 1500 in 75 mM HEPES (Roche Diagnostics, catalog number: 10783641001 )
Phosphate buffered saline 10x (Sigma-Aldrich, catalog number: P5493 )
Pierce 16% Formaldehyde (w/v), Methanol-free (Thermo Fisher Scientific, catalog number: 28908 )
30.Water, Molecular Biology Reagent (Sigma-Aldrich, catalog number: W4502 )
ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36970 )
TWEEN® 20 (Sigma-Aldrich, catalog number: P7949 )
Tris (Carl Roth, catalog number: 4855.2 )
Hoechst 34580 (Sigma-Aldrich, catalog number: 63493 )
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
Sodium Chloride (NaCl), BioXtra (Sigma-Aldrich, catalog number: S7653 )
Hydrochloric acid 36.5-38.0% (HCl), for molecular biology (Sigma-Aldrich, catalog number: H1758 )
DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (see Recipes)
PBS containing 0.1% gelatin (see Recipes)
10x TBS (see Recipes)
TBST (see Recipes)
4% Formaldehyde in 1x PBS (see Recipes)
Hoechst 34580 stock solution (1 mg/ml) (see Recipes)
TBST containing 0.25 μg/ml Hoechst 34580 (see Recipes)
Equipment
Hemocytometer
Timer
Fume hood
Cell culture hood (Thermo Fisher Scientific, model: HerasafeTM KSP , type KSP 12)
Fine tip curved tweezers
P10 pipettor (VWR, catalog number: 613-5259 )
P20 pipettor (VWR, catalog number: 613-5260 )
P200 pipettor (VWR, catalog number: 613-5263 )
P1000 pipettor (VWR, catalog number: 613-5265 )
Mini vacuum pump, KNF (A. Hartenstein, catalog number: AP86 )
Fluid aspiration system (VACCUBRAND, model: VHCpro )
CO2 incubator (Thermo Fisher Scientific, model: HeracellTM 150i )
Vortex mixer (VWR, catalog number: 444-1372 )
Pipette controller (accu-jet® pro, Brand, catalog number: 26300 )
-20 °C freezer
Refrigerator
Inverted microscope (Motic, model: AE31 )
Confocal laser-scanning microscope (ZEISS, model: LSM 780 )
Software
Fiji (ImageJ Version 2.0.0-rc-43/1.51h or more recent)
Microsoft Excel (version 14.6.7 or more recent)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
McNicoll, F. and Müller-McNicoll, M. (2018). A Quantitative Heterokaryon Assay to Measure the Nucleocytoplasmic Shuttling of Proteins. Bio-protocol 8(17): e2472. DOI: 10.21769/BioProtoc.2472.
Botti, V., McNicoll, F., Steiner, M. C., Richter, F. M., Solovyeva, A., Wegener, M., Schwich, O. D., Poser, I., Zarnack, K., Wittig, I., Neugebauer, K. M. and Müller-McNicoll, M. (2017). Cellular differentiation state modulates the mRNA export activity of SR proteins. J Cell Biol 216(7): 1993-2009.
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Category
Molecular Biology > Protein > Protein shuttling
Cell Biology > Cell-based analysis > Nucleocytoplasmic shuttling
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2,473 | https://bio-protocol.org/exchange/protocoldetail?id=2473&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Measurement of Dopamine Using Fast Scan Cyclic Voltammetry in Rodent Brain Slices
MM Madelyn I. Mauterer*
PE Paige M. Estave*
KH Katherine M. Holleran
Sara R. Jones
*Contributed equally to this work
Published: Vol 8, Iss 19, Oct 5, 2018
DOI: 10.21769/BioProtoc.2473 Views: 9823
Original Research Article:
The authors used this protocol in Jan 2018
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Jan 2018
Abstract
Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that allows sub-second detection of oxidizable chemical species, including monoamine neurotransmitters such as dopamine, norepinephrine, and serotonin. This technique has been used to record the physiological dynamics of these neurotransmitters in brain tissue, including their rates of release and reuptake as well as the activity of neuromodulators that regulate such processes. This protocol will focus on the use of ex vivo FSCV for the detection of dopamine within the nucleus accumbens in slices obtained from rodents. We have included all necessary materials, reagents, recipes, procedures, and analyses in order to successfully perform this technique in the laboratory setting. Additionally, we have also included cautionary points that we believe will be helpful for those who are novices in the field.
Keywords: Electrochemistry Voltammetry FSCV Dopamine Rodent Slice physiology
Background
Since the ability to examine the electrical properties of physiological systems was first appropriated for use in preclinical scientific research, many techniques used to study synaptic physiology have been developed. From the early days of electrophysiological recordings in squid axons to present day fast scan cyclic voltammetry (FSCV) performed in human Parkinson’s patients (Kishida et al., 2016; Lohrenz et al., 2016), the field has made significant advances in a relatively short amount of time. This protocol’s focus, FSCV, is the technical result of over 40 years of innovation and collaboration between physicists, analytical chemists, and neuroscientists. While electrochemistry was born with Michael Faraday and Alessandro Volta as early as the 19th century (Bard and Zoski, 2000), modern voltammetry did not come to fruition until the 1920s with Jaroslav Heyrovsky in his quest to measure the surface tension of mercury (Heyrovsky, 1922). Through his pursuit, Heyrovsky developed a dropping mercury electrode to perform polarography. This technique would be introduced as "voltammetry" in the United States in the 1940s and utilized platinum, gold, or carbon electrodes, in addition to the dropping mercury electrode, to study metal ions in solution. With the advent of computing technology, voltammetry methodology advanced dramatically from the late 1960s to today (for review see Bard and Zoski, 2000). Of note, in the 1970s, Ralph Adams pioneered the use of voltammetry, using a fast scanning method, in translational neuroscience specifically to study oxidizable neurotransmitters (Adams, 1976), a technique further applied to awake, freely-moving animals by Mark Wightman (Bucher and Wightman, 2015).
FSCV is a powerful electrochemical technique and is currently the only method available to directly measure extracellular levels of neurotransmitters on a sub-second timescale in discrete brain regions. One of the few comparable techniques is in vivo microdialysis–a method used to examine extracellular levels of multiple different neurotransmitters. However, even with the most recent advancements, microdialysis can only resolve neurotransmitter levels on a timescale of minutes, whereas FSCV has a temporal resolution of milliseconds. Other electrophysiological techniques utilize indirect measurements of neurotransmitter activity such as downstream postsynaptic ion channel-induced alterations in electrical signaling as a proxy. FSCV offers the unique ability to directly measure neurotransmitters in the extracellular space. This is due to the oxidizable nature of various chemical species, such as the monoamines dopamine, serotonin, and norepinephrine.
Since there are numerous publications regarding the fundamental theory of FSCV (for detailed review see: Yorgason et al., 2011; Rodeberg et al., 2017), we will not concentrate heavily on this topic here. Briefly, FSCV functions by passing an electrical current through an electrode implanted with a conductive substance such as carbon (referred to as the recording electrode), which receives electrochemical signals from a second stimulating electrode. More specifically, upon brief tissue stimulation by a bipolar stimulating electrode, dopamine is released into the extracellular space, which comes into contact with the recording electrode. A triangular waveform is passed within the carbon fiber of the electrode, ramping up to 1.2 m/sec and back down to -0.4 m/sec to detect dopamine, for example. In this way, when dopamine interacts with the carbon fiber at this specific command voltage, it rapidly oxidizes into dopamine-o-quinone, and reduces back into dopamine, which results in a signal that is communicated to the computing software. This results in the generation of a dopamine “trace” that can be modeled by the experimenter using Michaelis-Menten kinetics.
While the FSCV technique spans both in vivo and ex vivo applications, this protocol will specifically focus on FSCV execution in rodent brain slices. We will concentrate on ex vivo methods, as an analysis of current literature indicates there are few ex vivo FSCV protocols in rodent brain tissue–particularly using the new, freely available Demon Voltammetry Software (Maina et al., 2012; Fortin et al., 2015). While there are many reviews available regarding the history and theory of both in vivo and ex vivo FSCV, training in the execution of this technique in translational neuroscience is traditionally passed from mentor to mentee through direct hands-on training, with equipment often unique to each laboratory, rather than by formal instruction universal to all. Furthermore, until recently, commercial kits for FSCV were unavailable, and knowledge of these kits is still not widespread. Thus, it is imperative that trainees in the technique of FSCV be well versed in equipment usage and maintenance as well as technical performance. To this end, this protocol seeks to address technical execution of FSCV while directing the user to the tools and equipment the authors personally use to conduct experiments.
This protocol’s goal is to focus on rodent brain slice preparation, isolating a monoamine response (with a focus on dopamine in the nucleus accumbens), and data analysis. We also include a section with what we believe are helpful notes on the technique obtained from personal execution.
Brief Summary of Fast Scan Cyclic Voltammetry Procedure:
Production of carbon fiber electrodes.
Preparation Krebs stock solution and ACSF solution.
Brain extraction and brain slicing using a vibratome.
Isolation of an oxidizable neurotransmitter via stimulation of terminal fields using the fast scan cyclic voltammetry setup and Demon Voltammetry software.
Application of pharmacological agents or variation of stimulation parameters to study desired neurotransmitter dynamics.
Calibration of carbon fiber electrodes to identify electrode sensitivity to studied neurotransmitter.
Model neurotransmitter signals via Michaelis-Menten fitting to obtain various kinetic parameters,using Demon Voltammetry Analysis software.
Data exportation from Demon Voltammetry Analysis software to Microsoft Excel Spreadsheet.
Statistical analysis using program of choice(i.e., GraphPad Prism).
Materials and Reagents
Transfer Pipette, wide bore (Globe Scientific, catalog number: 135040 )
Stir bar (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F37122-0060 )
Insulin Syringe, 28 Gauge (Fisher Scientific, catalog number: 14-826-79)
Manufacturer: BD, catalog number: 329461 .
Borosilicate Capillary Glass with Microfilament, 1.2 mm x 0.68 mm, 4” (A-M Systems, catalog number: 602000 )
Carbon Fiber (GoodFellow, catalog number: C 005722 )
Stainless steel conductive wire (L 3,000 x 1,000 s x 1,000 s UL 1423, UL1423 30/1 BLU) with insulated segment (Kauffman Engineering, custom made)
Platinum wire, 0.5 mm dia, annealed (Alfa Aesar, catalog number: 43288 )
1 mm dia x 4 mm Ag/AgCl reference electrode (pellet form) (World Precision Instruments, catalog number: EP1 )
Bipolar Stimulating Electrode (Plastics One, catalog number: 8IMS3033SPCE )
Loctite® 404TM Instant Adhesive (VWR, catalog number: 300001-033)
Manufacturer: Henkel, Loctite® Professional Super Glue, catalog number: 442-46548 .
Ultrapure Water
Deionized Water
Isoflurane (Patterson Veterinary Supply, catalog number: 140430704 )
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6014 )
Sodium phosphate monobasic monohydrate (NaH2PO4•H2O) (Fisher Scientific, catalog number: S369-500 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
Magnesium chloride Hexahydrate (MgCl2•6H2O) (VWR, catalog number: BDH9244 )
D-(+)-Glucose (C6H12O6) (Sigma-Aldrich, catalog number: G8270 )
Calcium chloride dihydrate (CaCl2•2H2O) (Sigma-Aldrich, catalog number: C5080 )
L-ascorbic acid (C6H8O6) (Sigma-Aldrich, catalog number: A0278 )
Dopamine hydrochloride (Sigma-Aldrich, catalog number: H8502 )
Perchloric Acid (HClO4), ACS reagent, 60% (Sigma-Aldrich, catalog number: 311413 )
70% Ethanol solution (Fisher Scientific, catalog number: BP8201500 )
Artificial cerebrospinal fluid (ACSF) (see Recipes)
10x Krebs stock solution (see Recipes)
1 mM dopamine stock solution (see Recipes)
Equipment
100 ml plastic beaker (Cole-Parmer, catalog number: EW-06020-03 )
1 L plastic bottle (Cole-Parmer, catalog number: EW-06058-85 )
-20 °C freezer
Light source (Fisher Scientific, catalog number: 12-562-21 )
Acrylic Coronal Brain Matrix (Harvard Apparatus, catalog number: 62-0047 [for rat], 62-0050 [for mouse])
Personna Double Edge Stainless Razor Blade (Electron Microscopy Sciences, Personna, catalog number: 72000 )
Silver Print II (GC Electronics, catalog number: 22-023 )
Scissors (Harvard Apparatus, catalog number: 72-8422 )
Bone Rongeurs (Harvard Apparatus, catalog number: 72-8906 )
SpoonulaTM Lab Spoon (Fisher Scientific, catalog number: 14-375-10 )
Forceps (Fisher Scientific, catalog number: 10-300 )
Scalpel (Harvard Apparatus, catalog number: 72-8350 )
No. 13 Scalpel blade (Harvard Apparatus, catalog number: 72-8366 )
Induction Chamber (Harvard Apparatus, catalog number: 60-5246 )
Guillotine (Harvard Apparatus, catalog number: 73-1918 )
Alligator clips (Mueller, catalog number: BU-34 )
Gas Dispersion Tube (Corning, catalog number: 39533-12C )
Medical Gas Tank, carbogen (Linde)
Gas Regulator (VWR, catalog number: 55850-444 )
Stirrer (Thermo Fisher Scientific, catalog number: HP88857100 )
Vertical Microelectrode Puller (NARISHIGE, catalog number: PE-22 )
House Vacuum Line
Semiautomatic Vibrating Blade Microtome (Leica Microsystems, model: VT1200 S )
Chem-Clamp potentiostat (Dagan, catalog number: CHEM-5-MEG )
Headstage (5 Megohms) (Dagan, catalog number: 8024 )
Breakout Box, custom made (visit pineresearch- WaveNeuro Fast-Scan CV Potentiostat for FSCV bundles offered by PINE research. Many of their bundle options include the breakout box)
High-speed Analog Output Card (National Instruments, catalog number: PCI-6711 )
Data acquisition card (National Instruments, catalog number: PCIe-6351 )
Current Stimulus Isolator (Digitimer, model: NL800A )
Temperature Controller TC-344C (Harvard Apparatus, catalog number: 64-2401 )
Peristaltic Pump P-70 (Harvard Apparatus, catalog number: 70-7000 )
Upright Light Microscope with Reticle (example of suitable, Olympus, model: BX53M and Microscope World, model: KR887 )
3-Axis Manual Micromanipulator (NARISHIGE, model: MM-3 )
Calibration System, custom made (see Figure 5A)
Tygon® Tubing (Sigma-Aldrich, catalog number: Z685666)
Manufacturer: Saint-Gobain, catalog number: AJK00022 .
Syringe, 5 ml (Sigma-Aldrich, catalog number: Z116866 )
Syringe, 30 ml (Sigma-Aldrich, catalog number: Z683671 )
T connector with stopcock (Cole-Parmer, catalog number: UX-30600-02 )
Single Syringe Infusion Pump (Fisher Scientific, catalog number: 14-831-200 )
Fume hood (optional)
Superfusion chamber (Custom Scientific, custom order)
Clearlink System extension set with Control-A-Flo Regulator (Baxter, catalog number: 2C8891 )
Software
Demon Voltammetry and Analysis Software Suite, available free-of-charge at the following site: https://www.wakeforestinnovations.com/technologies/demon-voltammetry-and-analysis-software/
Note: A request for a license has to be submitted in order to download.
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Mauterer, M. I., Estave, P. M., Holleran, K. M. and Jones, S. R. (2018). Measurement of Dopamine Using Fast Scan Cyclic Voltammetry in Rodent Brain Slices. Bio-protocol 8(19): e2473. DOI: 10.21769/BioProtoc.2473.
Siciliano, C. A., Saha, K., Calipari, E. S., Fordahl, S. C., Chen, R., Khoshbouei, H. and Jones, S. R. (2018). Amphetamine Reverses Escalated Cocaine Intake via Restoration of Dopamine Transporter Conformation. The Journal of Neuroscience 38(2): 484-497.
Download Citation in RIS Format
Category
Neuroscience > Cellular mechanisms > Synaptic physiology
Cell Biology > Cell signaling > Synaptic transmision
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2,474 | https://bio-protocol.org/exchange/protocoldetail?id=2474&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Detection of Internal Matrix Targeting Signal-like Sequences (iMTS-Ls) in Mitochondrial Precursor Proteins Using the TargetP Prediction Tool
Felix Boos*
Timo Mühlhaus*
Johannes M. Herrmann
*Contributed equally to this work
Published: Vol 8, Iss 17, Sep 5, 2018
DOI: 10.21769/BioProtoc.2474 Views: 7933
Reviewed by: Hassan Rasouli
Original Research Article:
The authors used this protocol in Apr 2018
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The authors used this protocol in:
Apr 2018
Abstract
Mitochondria contain hundreds of proteins which are encoded by the nuclear genome and synthesized in the cytosol from where they are imported into the organelle. Sorting signals encoded in the primary and secondary sequence of these proteins mediate the recognition of newly synthesized precursor proteins and their subsequent translocation through the mitochondrial TOM and TIM translocases. Proteins of the mitochondrial matrix employ aminoterminal matrix targeting signals (MTSs), also called presequences, that are necessary and sufficient for their import into mitochondria. In most cases, these MTSs are proteolytically removed from the mature part of precursor proteins subsequent to their translocation into the matrix. Recently, internal MTS-like sequences (iMTS-Ls) were discovered in the mature region of many precursor proteins. Although these sequences are not sufficient for matrix targeting, they strongly increase the import competence of precursors by supporting their interaction with mitochondrial surface receptors. Due to their similarity to N-terminal MTSs, these iMTS-Ls can be identified using mitochondrial targeting prediction tools such as TargetP which was initially trained to recognize MTSs. In this protocol we describe how TargetP can be used to identify iMTS-Ls in protein sequences.
Keywords: Internal matrix targeting-signal like sequence Mitochondria Protein folding Protein import Signal sequence Targeting signals TargetP Tom70
Background
Targeting signals allow the correct intracellular distribution of newly synthesized proteins. Proteins that are transported into their target compartment in a folded conformation often show complex signals that are displayed on the surface of their native, folded structure. These signals are difficult to recognize in the primary sequence since they only can be deciphered when the fold of the native proteins is known.
In contrast, proteins that are threaded through translocases in the membrane of their target compartments in an unfolded conformation typically employ N-terminal signals. This organization allows that protein translocation can commence before translation is completed (Wickner and Schekman, 2005). N-terminal targeting sequences include the signal sequences found on secretory proteins, the transit peptides of chloroplast proteins, the MTSs of mitochondrial proteins and the leader peptides that target bacterial proteins into or across the inner membrane.
Prediction algorithms were developed that recognize and interpret these N-terminal targeting sequences (Juncker et al., 2009). These algorithms can either search for defined characteristic features (such as helicity, hydrophobicity, charge distribution or the presence of specific residues) or they are based on self-learning algorithms that were trained with test sets of proteins of known cellular localizations. One of the latter programs is TargetP which uses the primary sequence of a given protein (in FASTA or text format) to come up with a ‘probability’ score that predicts how likely it is that this sequence starts with a functional matrix targeting sequence (Emanuelsson et al., 2000; Emanuelsson et al., 2007). In the mitochondrial community, TargetP values of larger than 0.6 are typically interpreted as evidence that a given protein is targeted into the mitochondrial matrix.
Recent studies suggested that the important targeting information is not confined to the N-terminal MTS. Obviously, mature parts of proteins are critical for the translocation efficiency in vivo (Yamamoto et al., 2009; Chatzi et al., 2017; Backes et al., 2018). It is known for a long time that strongly folded domains can impede translocation (Eilers and Schatz, 1986; Harner et al., 2011; Schneider, 2018). Hence the unfolding probabilities of cytosolic domains of translocation intermediates strongly influence the velocity and yield of the translocation reaction (Wilcox et al., 2005; Yagawa et al., 2010). On the mitochondrial surface, protein unfolding is supported by cytosolic chaperones and by surface receptors, in particular by Tom70 (Hines et al., 1990; Söllner et al., 1990). The cytosolic domain of Tom70 represents a tetratricopeptide repeat structure which it shares with many co-chaperones of the Hsp90 and Hsp70 chaperone systems (Melin et al., 2015). Indeed, Tom70 cooperates with Hsp90 (in mammals) and Hsp70 (in mammals and fungi) in protein translocation across the outer membrane (Young et al., 2003; Bhangoo et al., 2007; Hoseini et al., 2016; Zanphorlin et al., 2016). It was recently shown that internal sequence stretches that share the typical biochemical properties of mitochondrial presequences support the binding to Tom70 and facilitate protein translocation into mitochondria (Backes et al., 2018, Hansen et al., 2018). Due to their similarity to mitochondrial targeting signals, these internal MTS-like sequences (iMTS-Ls) can be found using the same algorithms that are predicting mitochondrial presequences. The only necessary change one has to make is to relieve the restriction of analyzing only the N-terminus of a given protein, but iterate through the complete amino acid sequence. In this protocol, we explain how TargetP (or a similar tool) can be used to detect and analyze these iMTS-Ls protein sequences of interest. This will be helpful to analyze the features of mature regions of mitochondrial proteins and to estimate their import competence using a simple in silico test. We provide step-by-step instructions for three alternatives that contrast the different settings in data analysis. Depending on linking and scientific background, the detection of iMTS-Ls can be performed using either spreadsheet software (e.g., Microsoft Excel), a language and environment for statistical computing (R) or a general-purpose programming language (F#). While dedicated analysis platforms are the common choice in the field there is an advent for easy and intuitive general-purpose languages to facilitate the integration of the data analysis tasks into production tools and web apps or smooth the linkage of processing with high computational demand and statistical analysis.
Equipment
Computer or laptop with Internet access
Note: It should work for all common operating systems. However, certain restrictions might apply for specific operating systems. For a standard analysis, any contemporary hardware setup (desktop or notebook) should be suited for a reasonably fast result.
Software
In order to use the scripts provided within this protocol, at least one of the following software packages has to be installed:
Microsoft Excel or R
A spreadsheet such as Microsoft Excel, F# (https://fsharp.org/) with the libraries FSharp.Plotly and Fsharp.Stats, or R (www.r-project.org) with its packages seqinr, signal and Biostrings (Pagès et al., 2018).
TargetP 1.1 Server or TargetP package
The TargetP algorithm (Emanuelsson et al., 2007) can be used via the interface at the TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/). Alternatively, the TargetP package can be downloaded and used offline. See the instructions as described here: http://www.cbs.dtu.dk/services/TargetP/instructions.php. For the procedure explained here in this protocol, the online version is sufficient and the installation of the stand-alone software package is not required. However, this stand-alone package offers the possibility to assess large data sets (for example a genome-wide analysis) which is hardly possible with the online service.
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Boos, F., Mühlhaus, T. and Herrmann, J. M. (2018). Detection of Internal Matrix Targeting Signal-like Sequences (iMTS-Ls) in Mitochondrial Precursor Proteins Using the TargetP Prediction Tool. Bio-protocol 8(17): e2474. DOI: 10.21769/BioProtoc.2474.
Backes, S., Hess, S., Boos, F., Woellhaf, M. W., Godel, S., Jung, M., Muhlhaus, T. and Herrmann, J. M. (2018). Tom70 enhances mitochondrial preprotein import efficiency by binding to internal targeting sequences. J Cell Biol 217(4): 1369-1382.
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Category
Biochemistry > Protein > Structure
Cell Biology > Organelle isolation > Mitochondria
Biochemistry > Protein > Structure
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2,475 | https://bio-protocol.org/exchange/protocoldetail?id=2475&type=0 | # Bio-Protocol Content
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Peer-reviewed
Trace Fear Conditioning: Procedure for Assessing Complex Hippocampal Function in Mice
VS Vijendra Sharma
NC Noah Cohen
RS Rapita Sood
HO Hadile Ounallah-Saad
SG Shunit Gal-Ben-Ari
KR Kobi Rosenblum
Published: Vol 8, Iss 16, Aug 20, 2018
DOI: 10.21769/BioProtoc.2475 Views: 8772
Original Research Article:
The authors used this protocol in Jan 2018
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Jan 2018
Abstract
The trace fear conditioning protocol is designed to measure hippocampal function in mice. The protocol includes a neutral conditioned stimulus (tone) and an aversive unconditioned stimulus (shock), separated in time by a trace interval. The trace interval between the tone and the shock critically involves the hippocampus and could be used to evaluate hippocampal-dependent learning and memory. In this protocol, we presented mice with five pairings of tone and shock separated by a 20 sec trace interval. Freezing was measured 24 h after conditioning to evaluate contextual memory by placing mice in the conditioned chamber. In addition, 48 h after conditioning, freezing was measured in a dark chamber, which served as a different context. This method enables precise detection of hippocampal-dependent learning and memory following pharmacological and genetic manipulations that impair or enhance hippocampal function.
Keywords: Trace fear conditioning (TFC) Contextual memory Hippocampus function Memory enhancement Learning and memory deficits
Background
The trace fear conditioning (TFC) paradigm differs from standard fear conditioning paradigms (Heise et al., 2017; Segev et al., 2013 and 2015) by the simple insertion of a trace interval between a conditioned stimulus (CS, e.g., tone) and an unconditioned stimulus (US, e.g., electric foot shock), and repeated application of their combination at fixed intervals. The TFC paradigm involves the formation of temporally non-contiguous associations in both natural and pathological conditions, and is considered a complex, hippocampal-dependent paradigm, in contrast to simple cortical-dependent learning paradigms such as taste learning (Stern et al., 2013; Ounallah-Saad et al., 2014; Rappaport et al., 2015; Levitan et al., 2016; Sharma et al., 2018). A remarkable aspect of trace fear conditioning is that it provides a reliable model of attention-dependent associative learning that reflects the complex processing of the hippocampus and alters the circuitry recruited for learning. Several studies have shown that hippocampal lesions before and after training impair the ability of the animal to associate the CS and US stimuli when they are separated by the trace interval (Bangasser et al., 2006; Esclassan et al., 2009). However, animals with hippocampal lesions could associate the CS and US in a delay situation, where no trace interval separates them, but the CS and US co-terminate (McEchron et al., 1998; McEchron et al., 2000; Quinn et al., 2002). Although other brain regions such as the medial prefrontal cortex (Peters et al., 2009; Beeman et al., 2013), the entorhinal and perirhinal cortices (Esclassan et al., 2009; Kent and Brown, 2012), and the amygdala (Pape and Pare, 2010; Gilmartin et al., 2012) are involved in relaying stimulus inputs and response outputs, the hippocampus is selectively involved in trace conditioning rather than general fear learning or expression. Moreover, the acquisition of trace fear conditioning increases intrinsic excitability and facilitates LTP in pyramidal neurons of the hippocampus (Song et al., 2012), which makes trace fear conditioning an ideal paradigm to test hippocampal function in young and aged mice (Sharma et al., 2018).
Materials and Reagents
Animals
Male C57BL/6 mice (Envigo, Jerusalem) weighing 20-25 g and approximately 12 weeks old were used in this study. This protocol can also be used to study the function of the hippocampus in other strains and different age groups of mice (Shoji et al., 2016; Sharma et al., 2018). The mice were housed individually, on a 12/12 h light/dark cycle, and provided with water and standard rodent chow ad libitum. Animals were handled according to approved protocols and animal welfare regulations of the University of Haifa Institutional Ethics Committee.
70% ethanol (Fisher Scientific, catalog number: BP82011 )
Equipment
TFC chambers (Coulbourn Instruments, model: H10-11M-TC )
Place TFC chambers measuring 25 x 25 x 25 cm internally inside a larger, insulated plastic cabinet that excludes external light and noise (Panlab, Harvard Apparatus, model: LE116 76-0280 ).
Visual (CCD) and infrared camera (Sensor Technologies America, model: STC-CMB4MPOE ) along with an infrared illuminator (Bosch, model: EX12LED-3BD-8W ).
Software
FreezeFrame 3.0 and FreezeView software (Coulbourn Instruments)
Note: Both software components can be downloaded from the Actimetrics website.
FreezeView manual
Note: The manual can be downloaded from the Coulbourn webpage.
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Sharma, V., Cohen, N., Sood, R., Ounallah-Saad, H., Gal Ben-Ari, S. and Rosenblum, K. (2018). Trace Fear Conditioning: Procedure for Assessing Complex Hippocampal Function in Mice. Bio-protocol 8(16): e2475. DOI: 10.21769/BioProtoc.2475.
Sharma, V., Ounallah-Saad, H., Chakraborty, D., Hleihil, M., Sood, R., Barrera, I., Edry, E., Kolatt Chandran, S., Ben Tabou de Leon, S., Kaphzan, H. and Rosenblum, K. (2018). Local inhibition of PERK enhances memory and reverses age-related deterioration of cognitive and neuronal properties. J Neurosci 38(3): 648-658.
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Category
Neuroscience > Behavioral neuroscience > Learning and memory
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2,478 | https://bio-protocol.org/exchange/protocoldetail?id=2478&type=0 | # Bio-Protocol Content
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This is a correction notice. See the corrected protocol.
Peer-reviewed
Correction Notice: Strategies for Performing Dynamic Gene Perturbation Experiments in Flowers
Diarmuid S. Ó’Maoiléidigh
EG Emmanuelle Graciet
FW Frank Wellmer
Published: Oct 5, 2017
DOI: 10.21769/BioProtoc.2478 Views: 2095
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The first author of this protocol was mistakenly listed as "Emmanuelle Graciet" in the previous version of this protocol (https://bio-protocol.org/e1774).It should be "Diarmuid S. Ó’Maoiléidigh". It has been corrected in the current version of this protocol. Bio-protocol apologizes for this error.
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How to cite：Ó’Maoiléidigh, D. S., Graciet, E. and Wellmer, F. (2017). Correction Notice: Strategies for Performing Dynamic Gene Perturbation Experiments in Flowers . Bio-protocol 7(19): e2478. DOI: 10.21769/BioProtoc.2478.
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248 | https://bio-protocol.org/exchange/protocoldetail?id=248&type=0 | # Bio-Protocol Content
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RNA Extraction from RNase-Rich Senescing Leaf Samples
Susheng Gan
Published: Vol 2, Iss 16, Aug 20, 2012
DOI: 10.21769/BioProtoc.248 Views: 11297
Original Research Article:
The authors used this protocol in Apr 1987
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Apr 1987
Abstract
Isolation of intact, full-length high quality RNAs is essential for RNA sequencing, reverse transcription PCR analysis of gene expression as well as RNA gel blot analysis. This simple yet easy protocol is developed to meet this need; in addition to regular samples, this protocol is especially good for isolating RNAs from RNase-rich samples such as senescing leaves and ripening fruits (from which RNAs isolated using standard method are generally degraded to certain degree). The total RNA yield varies from 900 μg total RNA/g non-senescing leaves to 200 μg total RNA/g senescent leaves.
Keywords: RNA Senescence Leaf Arabidopsis
Materials and Reagents
Guanidinium thiocyanate (Thermo Fisher Scientific, catalog number: BP221 )
NaCitrate
N-Lauroylsarcosine (Sigma-Aldrich, catalog number: L9150 )
β-mercaptoethanol (β-ME)
Glacial HAc
NaOH
Phenol (J.T.Baker®, catalog number: 2859 )
Ethylenediaminetetraacetic acid (EDTA)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
Tris (hydroxymethyl) aminomethane
Chloroform
Isopropanol
Diethylpyrocarbonate (DEPC)
Ethanol
Extraction buffer (EB) (see Recipes)
2 M NaAcetate (pH 4.0) (see Recipes)
2 M NaAcetate (pH 5.0) (DEPC treated) (see Recipes)
H2O-saturated phenol (no buffer required) (see Recipes)
Citrate-EDTA-SDS solution (CES) (see Recipes)
Tris-EDTA-SDS solution (TES) (see Recipes)
100 ml 0.75 M NaCitrate (see Recipes)
60 ml 10% Sarkosyl (see Recipes)
Equipment
Vortex Mixer
Beckman high speed centrifuge with JS13.1 rotor or the like
15-ml Corning centrifuge tube
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Gan, S. (2012). RNA Extraction from RNase-Rich Senescing Leaf Samples. Bio-protocol 2(16): e248. DOI: 10.21769/BioProtoc.248.
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Category
Plant Science > Plant molecular biology > RNA
Molecular Biology > RNA > RNA extraction
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249 | https://bio-protocol.org/exchange/protocoldetail?id=249&type=0 | # Bio-Protocol Content
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Peer-reviewed
Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC)
RS Roman Sobotka
LK Luděk Kořený
JK Jana Kopečná
MO Miroslav Oborník
Published: Vol 2, Iss 16, Aug 20, 2012
DOI: 10.21769/BioProtoc.249 Views: 9222
Original Research Article:
The authors used this protocol in Sep 2011
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Abstract
This is an accurate method to assess the rate of chlorophyll biosynthesis in vivo in cyanobacteria. Given that labeled glutamate is used as the very early precursor of chlorophyll together with a short pulse of labeling (30 min), this method provides information about the metabolic flow through the whole chlorophyll biosynthetic pathway on a short timescale.
Keywords: Chlorophyll biosynthesis 14C radiolabeling Cyanobacteria Synechocystis Thin Layer Chromatography
Materials and Reagents
Synechocystis PCC 6803
Glutamic acid [U-C14] (ARC 0165A, American Radiolabeled Chemicals) ([14C]Glu)
Methanol
25% ammonia solution
1 M NaCl
Hexane
10% KOH
Petroleum ether
Chloroform
1 M Na2HPO4
1 M NaH2PO4
X-ray film (Eastman Kodak Company)
NH4OH
1 M TES [2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid]/NaOH (pH 8.2)
Growth medium BG11
Equipment
10 ml Headspace vials (Sigma-Aldrich)
Water bath shaker
2 ml o-ring cap tubes
Glass beads (100-200 μm)
Vortex
Tabletop centrifuge (MiniSpin plus, Eppendorf)
Speedvac Concentrator plus (Eppendorf)
Silica gel TLC plate (SIL G-25, MACHEREY-NAGEL)
Rectangular TLC developing tank (Sigma-Aldrich)
Mikro 22R centrifuge (Hettich)
MiniSpin centrifuge (Eppendorf)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sobotka, R., Kořený, L., Kopečná, J. and Oborník, M. (2012). Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC). Bio-protocol 2(16): e249. DOI: 10.21769/BioProtoc.249.
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Category
Biochemistry > Other compound > Chlorophyll
Microbiology > Microbial biochemistry
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250 | https://bio-protocol.org/exchange/protocoldetail?id=250&type=0 | # Bio-Protocol Content
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Co-immunoprecipitation in Yeast
Olesya O. Panasenko
Published: Vol 2, Iss 16, Aug 20, 2012
DOI: 10.21769/BioProtoc.250 Views: 21976
Original Research Article:
The authors used this protocol in Feb 2012
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Abstract
This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically binds to that particular protein (antigene protein). The partner proteins that are bound to a specific target protein can be co-immunoprecipitated together with an antigen. These protein complexes can then be analyzed to identify new binding partners, binding affinities, the kinetics of binding and the function of the target protein. Here I describe the protocols that allow to immunoprecipitate different protein complexes, for example NAC complex (Panasenko et al., 2009), Ccr4-Not complex (Panasenko and Collort, 2011), ribosomes (Panasenko and Collort, 2012) and investigate their partners. For each CoIP I used the different lysis buffer, as indicated below in recipes.
Keywords: Immunoprecipitation Protein interaction Affinity interaction Antibody Yeast
Materials and Reagents
Glass beads for cells breaking 0.5 mm (Bio Spec Products, catalog number: 110/9105 )
Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 ) solution 100 mg/ml prepared on ethanol.
Bradford reactive (Bio-Rad Laboratories, catalog number: 500-0006 ).
Different types of the beads can be used depending on the particular antigene protein and antibodies.
Protein G magnetic Dynabeads (Life Technologies, Invitrogen™, catalog number: 100.04D ) or Protein A magnetic Dynabeads (Life Technologies, Invitrogen™, catalog number: 100.02D )
Protein G Sepharose (Amersham biosciences, catalog number: 17-0618-01 ) or Protein A Sepharose (Amersham biosciences, catalog number: 17-0780-01 )
Magnet, in case of using magnetic beads (Life Technologies, Invitrogen™, catalog number: 123.21D )
Antibodies
For example Peroxidise-anti-peroxidase soluble complex (PAP-antibodies) were bought from Sigma-Aldrich (catalog number: P1291 ). Antibodies against HA (HA.11) (Clone 16B12, catalog number: MMS-101R ) and Myc [c-myc (9E10), catalog number: MMS-150R ] were bought from Covance.
Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 13560400 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7625 ) 100 mM solution prepared on isopropanol
Laemmli sample buffer
Lysis buffer for protein complexes IP (see Recipes)
Lysis buffer for ribosome IP (see Recipes)
Equipment
Table Centrifuges
Glass bead beater Genie Disruptor (Scientific Industries, catalog number： SI-DD38 )
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Panasenko, O. O. (2012). Co-immunoprecipitation in Yeast. Bio-protocol 2(16): e250. DOI: 10.21769/BioProtoc.250.
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Category
Biochemistry > Protein > Immunodetection
Microbiology > Microbial biochemistry > Protein
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2,501 | https://bio-protocol.org/exchange/protocoldetail?id=2501&type=0 | # Bio-Protocol Content
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Peer-reviewed
A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
JA Julia F. Alterman
AC Andrew H. Coles
LH Lauren M. Hall
NA Neil Aronin
AK Anastasia Khvorova
MD Marie-Cécile Didiot
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2501 Views: 9997
Edited by: Longping Victor Tse
Reviewed by: Ehsan KheradpezhouhSébastien Gillotin
Original Research Article:
The authors used this protocol in Oct 2016
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Oct 2016
Abstract
Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo. We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.
Keywords: Primary cortical neurons siRNA Screening Branched DNA QuantiGene 2.0
Background
Oligonucleotide therapeutics represent a new class of drug that can target any genetically defined disorder, by silencing the expression of mutant proteins. Specifically, siRNAs are double stranded oligonucleotides that are loaded into the RNA induced silencing complex (RISC) and can silence mRNA before it is translated. However, unmodified siRNAs are unstable and cannot enter cells without the help of cationic lipid formulation, which can be toxic to primary cells such as neurons. In this protocol, we use self-delivering, hydrophobically modified siRNAs (hsiRNAs) for mRNA silencing. Recent progress in the chemistry of oligonucleotides has enabled the design of these stabilized hsiRNAs, which promote cellular internalization, efficient entry into RISC, and potent knockdown of target genes (Byrne et al., 2013; Alterman et al., 2015; Ly et al., 2017). These compounds contain 2’-O-methyl and 2’-fluoro modifications on all sugars and phosphorothioate backbone modifications; the oligonucleotides are often conjugated to a hydrophobic moiety, such as cholesterol, to support membrane binding and cellular internalization without toxicity. This new class of compounds offers researchers a straightforward method for silencing various genes in the context of biologically relevant, and hard to transfect, primary cortical neurons (Alterman et al., 2015).
Today, in the early stage of drug discovery, most high-throughput tests are performed in cell-based assays for lead oligonucleotide identification and validation. Cell-based assays improve and accelerate drug screening, providing more relevant in vivo biological information than biochemical assays and reducing the need for animal testing. High-throughput assays performed in primary neurons have emerged as a powerful tool to discover new therapies for the treatment of neurodegenerative disorders, such as Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) or Alzheimer’s disease (AD) (Sharma et al., 2012). Cell based assays using primary neurons provide a more natural (relevant) environment for studying neurodegenerative disorders than clonal neuronal lines. Transfecting oligonucleotide-based therapeutics into primary neurons generally relies on approaches such as electroporation (Mertz et al., 2002; Gresch et al., 2004; Zhang et al., 2016), viral transduction (Naldini et al., 1996; Hughes et al., 2002; Janas et al., 2006) or lipid-mediated transfection (Ohki et al., 2001; Dalby et al., 2004; Zhang et al., 2016). However, these methods can be laborious, show low efficiency, and induce cellular toxicity. Thus, the self-delivering properties of hsiRNAs represent an effective method to identify new leads in vitro in complex cellular models, such as primary neurons. We have recently demonstrated that hsiRNAs efficiently bind neuronal-cell membranes within seconds after treatment, enter cells, and induce potent gene silencing, both in vitro, in primary neurons, and in vivo, in mouse brain, all without the use of transfection reagents (Alterman et al., 2015; Ly et al., 2017).
Our laboratory has established a rapid high-throughput platform to identify and validate hsiRNA leads in primary neurons in vitro in 96-well format. To quantify the amount of target mRNA upon hsiRNA treatment, we use the QuantiGene 2.0 branched DNA (bDNA) assay, a high-throughput, 96-well plate-based mRNA quantification assay. This technique is designed to directly quantify the target mRNA from sample lysate without the need to purify the RNA, thus minimizing sample manipulation (Kern et al., 1996; Coles et al., 2015). This assay enables accurate and precise detection of even low abundance mRNAs, minimizing experimental variability and error (Collins et al., 1997; Canales et al., 2006). The combination of both self-delivering hsiRNAs and high-throughput quantification of mRNA accelerates the identification of efficacious oligonucleotides in primary neurons.
Here, we describe the workflow of this high throughput assay for performing large-scale screening and dose response validation of hsiRNAs in primary cortical neurons. We detail our methods for primary cortical neuron preparation in 96-well plate format (Figure 2), neuronal treatment with hsiRNA, assay plate management, mRNA quantification, and data analysis. This platform can be used for the screening of oligonucleotide therapeutics in primary neurons for the potential treatment of neurodegenerative diseases.
Materials and Reagents
Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516 )
Deep 96-well sterile polypropylene plate (Corning, Axygen®, catalog number: 391-04-062 )
Tips (from 0.2 μl to 1 ml) (VWR)
Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar)
Fire-polished Pasteur pipet with cotton plug (made in-house)
Tissue culture treated 10 cm dish (Corning, catalog number: 430167 )
50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352098 )
1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22-282 )
1 ml sterile syringe (BD, catalog number: 309659 )
Adhesive plate seals (VWR, catalog number: 60941-126 )
Pregnant wild-type mice (THE JACKSON LABORATORY)
Cholesterol-conjugated hsiRNAs (designed and produced in-house)
Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F )
Poly-L-lysine (Sigma-Aldrich, catalog number: P4707 )
Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV )
200-Proof ethanol (Decon Labs, catalog number: 2701 )
DMEM cell culture medium (Mediatech, catalog number: 10-013-CV )
Hibernate E (BrainBits, catalog number: HE )
DNase I (Worthington, catalog number: 54M15168 )
Papain (Worthington, catalog number: 54N15251 )
Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: QS0011 )
QuantiGene 2.0 Probe sets (varies by gene)
Lysis mixture (Thermo Fisher Scientific, AffymetrixTM, catalog number: 13228 )
Proteinase K (Thermo Fisher Scientific, AffymetrixTM, catalog number: QS0103 )
Neurobasal medium NbActiv4 (BrainBits, catalog number: Nb4-500 )
Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV )
NeuralQTM medium (Sigma-Aldrich, catalog number: N3100 ) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800 ), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185 )
5’UTP (Sigma-Aldrich, catalog number: U6625 )
5’FdU (Sigma-Aldrich, catalog number: F3503 )
Papain/DNase solution (see Recipes)
Plating medium (see Recipes)
Feeding medium (see Recipes)
Equipment
Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus )
Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus )
Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II, Type A2 )
Bunsen burner
Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V )
Water bath at 37 °C (Fisher Scientific, model: Model 2332 )
4 °C fridge
Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12 )
Set of two forceps (Fine Science Tools, catalog number: 11251-30 )
Dissection microscope (Motic, model: SMZ168 Series )
Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP )
Tissue culture phase-contrast inverted microscope (Motic, model: AE2000 )
Hemocytometer (0.1000-0.0025 mm2) (Neubauer)
µPlate carrier (Beckman Coulter, model: SX4750 )
Autoplate washer (BioTek Instruments, model: ELx405 )
Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R )
Allegra X-15R rotor (Beckman Coulter, model: SX4750 )
Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro )
Software
Microsoft Office Excel (Microsoft Office)
GraphPad Prism 7 software (GraphPad Software)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Alterman, J. F., Coles, A., Hall, L. M., Aronin, N., Khvorova, A. and Didiot, M. (2017). A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics. Bio-protocol 7(16): e2501. DOI: 10.21769/BioProtoc.2501.
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Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Neuroscience > Nervous system disorders > Animal model
Molecular Biology > RNA > RNA detection
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2,502 | https://bio-protocol.org/exchange/protocoldetail?id=2502&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions
AT Amit K. Tripathi
Ashwani Pareek
Sneh Lata Singla-Pareek
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2502 Views: 13564
Edited by: Marisa Rosa
Reviewed by: Yingnan Hou
Original Research Article:
The authors used this protocol in Aug 2016
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The authors used this protocol in:
Aug 2016
Abstract
DNA damage is one of the common consequences of exposure to various stress conditions. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues exposed to different stress agents. However, owing to the presence of firm cellulosic cell wall and phenolics, plant cells and tissues are not easily amenable to be subjected to these assays. Here, we describe an optimized TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay-based protocol to determine the extent of DNA fragmentation and programmed cell death in plant root cells subjected to various stress conditions. The method described here has the advantages of simplicity, reliability and reproducibility.
Keywords: DNA fragmentation Free DNA termini Genotoxic Programmed cell death (PCD) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
Background
Exposure to various stresses generally leads to at least some degree of DNA damage resulting in various lesions such as thymine dimerization, alkylation of bases, single stranded nicks, and double-stranded breaks (Bray and West, 2005; Manova and Gruszka, 2015). Of all types of DNA damage, DNA fragmentation is of particular concern during stress conditions, which may either be a direct effect of the stress (as observed, for example, upon treatment with genotoxic agents) or an indirect effect (predominantly, via excessive generation of reactive oxygen species) or may even be a cumulative consequence of both (Bray and West, 2005; Kapoor et al., 2015). This DNA damage must be accurately repaired by the cell’s repair machinery, failing which there may be deleterious consequences including cell death. For maintaining the normal state, cells utilize the DNA damage response which relies on three non-exclusive events viz. detection/recognition of the damage, its access by the repair machinery and finally its repair (Smerdon, 1991).
One of the major molecular mechanisms of stress adaptation at the cellular level involves the resistance to DNA damage and/or efficient repair of the damaged DNA caused due to stress. Therefore, to assess the stress adaptability of a genotype, accurate assessment of DNA damage is often needed. Two widely-used assays to detect DNA fragmentation in plants are Single Cell Gel Electrophoresis–also known as Comet assay (Santos et al., 2015), and TUNEL [Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling] assay. In comet assay, the tissue of interest is sliced and the resulting cell suspension containing nuclei is embedded in an agarose matrix followed by its alkaline electrophoresis and staining with DAPI/ethidium bromide. After electrophoresis, micrographs show the appearance of broken DNA like a tail similar to that of a comet while the undamaged and condensed DNA appears like a spherical mass forming the head of the comet (Wang et al., 2013). Comet assay, though quite useful, has a few limitations. For instance, it requires isolated nuclei, and hence gives no information on the distribution of DNA damage in a given tissue as well as regarding programmed cell death (PCD). The other widely-used assay–TUNEL assay, can be used to detect in situ DNA strand breaks. TUNEL assay is based on incorporation of labeled dUTP in the DNA (mediated by the enzyme terminal deoxynucleotidyl transferase) which occurs only at the regions with free 3’ termini (i.e., breaks or extreme ends of the chromosome) (Gavrieli et al., 1992). Besides, as breaks in inter-nucleosomal DNA often lead to programmed cell death, TUNEL assay provides significant information about PCD. TUNEL assay, in its basic form, also offers the advantages of simplicity and can give an idea about the distribution of DNA fragmentation (TUNEL-positive cells) in the tissue being studied.
Plant tissues are not easily amenable to some of the steps of TUNEL assay. The major reasons for this are: difficulty in permeabilization due to the presence of cellulosic cell wall and potential inhibition of TdT-catalyzed reaction by phenolics present in the plant cells. Due to these reasons, TUNEL assay is not a frequently utilized procedure for assessment of DNA fragmentation and PCD in plants. A few recent studies, nonetheless, have shown the application of TUNEL assay in rice (Kwon et al., 2013) and Arabidopsis (Phan et al., 2011; Yang et al., 2014). However, most of these studies have used microtomy/ultramicrotomy and ‘paraffin section’ preparation–a procedure which is not very easy, and requires somewhat expensive instrumentation and technical expertise. Given the range of information which TUNEL assay can provide, especially when determining the stress adaptability of plant genotypes, and its advantages in comparison to other methods, there is a need to develop a standardized, easy-to-follow and relatively inexpensive protocol for TUNEL assay using plant tissues.
Here, we describe an optimized TUNEL assay-based protocol to assess the extent of DNA fragmentation and programmed cell death in plant root cells under various stress conditions. The protocol presented here describes, in detail, a more generalized version of the methodology used for TUNEL assay in our recent study (Tripathi et al., 2016). While we often use this method to study DNA damage and PCD in root tissue from rice and Arabidopsis, it can also be utilized to study these phenomena in root tissue from other herbaceous plants with some minor modifications as detailed in the ‘Procedure’ section. The method presented here is quite easy-to-follow, reliable and reproducible.
Materials and Reagents
1-200 μl pipet tips (DNase-free) (Corning, USA)
0.2-2 μl pipet tips (DNase-free) (Corning, USA)
100-1,000 μl pipet tips (DNase-free) (Corning, USA)
Razor blade (any standard make)
1.5 ml and 2 ml microcentrifuge tubes (DNase-free) (Corning, USA)
15 ml and 50 ml centrifuge tubes (DNase-free) (Corning, USA)
Aluminium foil (any standard make)
Glass slides (any standard make)
Cover slips (any standard make)
1-4 weeks old rice (Oryza sativa cv. IR64) seedlings (see Note at the beginning of the ‘Procedure’ section)
Ethanol (Sigma-Aldrich, catalog number: 24102 )
Note: This product has been discontinued.
ProLong® Gold Antifade mountant with DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36931 )
DeadEndTM Fluorometric TUNEL System (Promega, catalog number: G3250 )
Note: *In case the DeadEndTM Fluorometric TUNEL System (Promega, catalog number: G3250 ) is being used, then the chemicals/reagents marked with an asterisk (*) need not be procured. See Note 2 below.
Sodium chloride (NaCl) (AMRESCO, catalog number: 0241 )
Potassium chloride (KCl) (AMRESCO, catalog number: 0395 )
Sodium phosphate dibasic (Na2HPO4) (AMRESCO, catalog number: 0404 )
Potassium phosphate monobasic (KH2PO4) (AMRESCO, catalog number: 0781 )
Paraformaldehyde powder (Sigma-Aldrich, catalog number: 158127 )
Citric acid monohydrate (Sigma-Aldrich, catalog number: C1909 )
Trisodium citrate dihydrate (Sigma-Aldrich, catalog number: S1804 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Tris(hydroxymethyl)aminomethane (Sigma-Aldrich, catalog number: 252859 )
Sodium cacodylate* (Sigma-Aldrich, catalog number: C4945 )
Cobalt(II) chloride hexahydrate* (Sigma-Aldrich, catalog number: C8661 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 10708984001 )
Manufacturer: Roche Diagnostics, catalog number: 10708984001 .
Bovine serum albumin (BSA) (AMRESCO, catalog number: 0332 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: 03620 )
Fluorescein-12-dUTP* (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0101 )
dATP* (Promega, catalog number: U1202 )
Terminal Deoxynucleotidyl Transferase, Recombinant (rTdT)* (Promega, catalog number: M1871 )*
Sodium citrate
Hydrochloric acid (HCl)
Propidium iodide (PI) (Sigma-Aldrich, catalog number: 81845 )
DNase-free proteinase K (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2544 )
1x phosphate buffered saline (PBS), pH 7.4 (see Recipes)
Fixative buffer (see Recipes)
100 mM citric acid solution (see Recipes)
100 mM trisodium citrate dihydrate solution (see Recipes)
100 mM sodium citrate buffer, pH 6.0 (see Recipes)
Permeabilization solution (see Recipes)
Proteinase K (see Recipes)
Equilibration buffer (see Recipes)
Nucleotide mix (see Recipes)
TUNEL reaction mix (see Recipes)
20x saline-sodium citrate (SSC) buffer (see Recipes)
2x saline-sodium citrate (SSC) buffer (see Recipes)
Propidium iodide stock solution (1 μg/μl) (see Recipes)
Equipment
0.2-2 μl pipette (Gilson, PIPETMAN Classic®)
2-200 μl pipette (Gilson, PIPETMAN Classic®)
100-1,000 μl pipette (Gilson, PIPETMAN Classic®)
Glass beakers of volume 20 ml, 50 ml, and 100 ml (Schott Duran, Germany)
Reagent bottles, glass of volume 50 ml, 100 ml, 500 ml, and 1,000 ml (any standard make)
Laboratory fume hood (any standard make)
Magnetic stirrer (Genetix Brand, India)
Water bath (any standard make)
Confocal microscope (Nikon, model: Nikon A1R )
Software
ImageJ Standard version 1.46r (http://imagej.net/mbf/)
NIS Elements AR (Nikon, Japan) or a comparable software for confocal microscope image acquisition and analysis
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Tripathi, A. K., Pareek, A. and Singla-Pareek, S. L. (2017). TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions. Bio-protocol 7(16): e2502. DOI: 10.21769/BioProtoc.2502.
Tripathi, A. K., Pareek, A. and Singla-Pareek, S. L. (2016). A NAP-family histone chaperone functions in abiotic stress response and adaptation. Plant Physiol 171(4): 2854-2868.
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Category
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > DNA labeling
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2,503 | https://bio-protocol.org/exchange/protocoldetail?id=2503&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Improved Oviduct Transfer Surgery for Genetically Modified Rat Production
LL Laura J. Lambert
LJ Larry W. Johnson
DK Daniel Kennedy
JC Joan Cadillac
Robert A. Kesterson
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2503 Views: 9361
Reviewed by: Jingli Cao
Original Research Article:
The authors used this protocol in 14-Oct 2013
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Original research article
The authors used this protocol in:
14-Oct 2013
Abstract
Rat embryo transfer surgeries are becoming more common with targeted nucleases increasing the demand for rat models. This protocol details pre-surgical preparation, improved surgical techniques for placing embryos into the oviduct, and post-surgical care of rats to parturition. Direct application of mouse oviduct transfer protocols results in limited success in the rat. By combining techniques from several widely used protocols in the field, increased yield of live pups born to healthy dams is possible. This protocol is distinct from previously published protocols by the use of a modified anesthesia protocol (Smith et al., 2004), use of analgesia, the addition of peritoneal sutures (Filipiak and Saunders, 2006), incision location and number of transfers per animal (Krinke et al., 2000).
Keywords: Rat Transgenics Embryos Surgery Oviduct
Background
The ability to reliably produce healthy pups after microinjection and embryo transfer surgery is critical to model creation and, in particular, the increased likelihood of creating multiple founder animals gives confidence in the phenotypes observed. Therefore, as birth rates were low relative to reported rates even with varied concentrations of injection solution, modifications were systematically made to the existing mouse embryo transfer protocol to better suit the rat.
Multiple publications describe transferring embryos to the oviducts of both horns of the bipartite uterus; however, this increases the length of time the animal is under anesthesia and requires either a midline incision and traversing the peritoneal cavity to reach the lateral reproductive tract, or creating two separate incisions (Krinke et al., 2000). These options are less than ideal since either will increase stress of the animal and thereby the likelihood that the pregnancy will be aborted. By creating a single lateral incision and administering analgesia both preoperatively and postoperatively the stress of the animal is minimized (Smith et al., 2004). The use of isoflurane over injectable anesthetic agents minimizes risk of toxicity (such as seen with tribromoethanol), injury from IP injection, and repeated dosing, all of which are associated with higher mortality rates following rodent surgery (Bernal et al., 2009).
The greatest improvements in litter number and size followed the addition of ampicillin and epinephrine to the procedure [62 born/298 transferred (20.8%) versus 91 born/248 transferred (36.7%) post addition of ampicillin and epinephrine; all projects]. Although the surgery is performed aseptically, ampicillin was shown as early as 1995 to optimize the number of pups born to rats (Waller, 1995) and use of epinephrine on the ovarian bursa reduced bleeding and thereby trauma to the animal, as well as reduced the length of time required to find the infundibulum. These modifications have been used individually in multiple reports; however, this is the first protocol to combine the most advantageous aspects of each protocol while refining procedures that may be detrimental (Krinke et al., 2000; Smith et al., 2004; Filipiak and Saunders, 2006).
Materials and Reagents
Personal protective materials–hair net, gloves
4-0 black silk sutures (Kent Scientific, catalog number: INS701073 )
9 mm wound clips (BD, catalog number: 427631 )
Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
Insulin syringes (BD, catalog number: 329412 )
Iodine swabs (PDI Healthcare, catalog number: S41350 )
(Optional) Rodent mask diaphragms (Smiths Medical, Surgivet, catalog number: 32247B1 )
Sterile surgical drape
500 ml filter system
Fertilized one cell Sprague Dawley embryos (see Notes)
8-week old female Sprague Dawley recipient rats
Vasectomized Sprague Dawley male rats
Luteinizing hormone releasing hormone agonist (LHRHa) (Sigma-Aldrich, catalog number: L4513 )
70% ethanol
Buprenorphine (Southern Anesthesia & Surgical, catalog number: 12496075705 )
Carprofen (Zoetis Services, catalog number: 060062 )
Ampicillin (Fisher Scientific, catalog number: BP1760-25 )
0.1% epinephrine (Acros Organics, catalog number: 204400010 )
Sterile, nonmedicated ophthalmic ointment (Rugby Laboratories, catalog number: 301905 )
Embryo tested water (Sigma-Aldrich, catalog number: W1503 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M1880 )
Glucose (Sigma-Aldrich, catalog number: 158968 )
Penicillin (Sigma-Aldrich, catalog number: P7794 )
Streptomycin (Sigma-Aldrich, catalog number: S1277 )
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
Sodium pyruvate (Sigma-Aldrich, catalog number: P4562 )
EDTA (Sigma-Aldrich, catalog number: 03609 )
L-Glutamine (Sigma-Aldrich, catalog number: G8540 )
Sodium lactate (Sigma-Aldrich, catalog number: L7900 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
Phenol red (Sigma-Aldrich, catalog number: P0290 )
Isoflurane (AMERISOURCE BERGEN, catalog number: 10103618 )
LHRHa solution (see Recipes)
KSOM medium (see Recipes) (Cold Spring Harbor, 2006)
Equipment
Biosafety cabinet
pH meter
Mouth pipet (Fisher Scientific, catalog number: NC9048719 )
Manufacturer: BIOTECH, model: MP001Y.
Glass pipettes (Fisher Scientific, catalog number: 13-678-20C )
Flame source to pull pipettes
Personal protective equipment–clean lab coat
9 mm wound clip applier (BD, catalog number: 427630 )
Microscope (Leica Microsystems, model: Leica S8 APO )
Fine forceps (Fine Science Tools, catalog number: 11251-10 )
Spring scissors (Roboz Surgical Instrument, catalog number: RS-5650 )
Large scissors (Roboz Surgical Instrument, catalog number: RS-5910 )
Grip forceps (Roboz Surgical Instrument, catalog number: RS-8100 )
Micro clip (Roboz Surgical Instrument, catalog number: RS-5420 )
Versi-Dry surface protectors (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 74000-00 )
37 °C warming plate (C & A Scientific, Premiere, catalog number: XH-2002 )
Animal clippers (Oster, catalog number: 078005-301-003 )
Bead sterilizer (CellPoint Scientific, catalog number: 5-1450 )
Anesthesia machine (Smiths Medical, Surgivet, catalog number: WWV9000 )
Rodent anesthesia circuit set (Smiths Medical, Surgivet, catalog number: V7103 )
Large rubber bands
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lambert, L. J., Johnson, L. W., Kennedy, D., Cadillac, J. and Kesterson, R. A. (2017). Improved Oviduct Transfer Surgery for Genetically Modified Rat Production. Bio-protocol 7(16): e2503. DOI: 10.21769/BioProtoc.2503.
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Category
Cell Biology > Cell Transplantation > Embryo Transplant
Cell Biology > Tissue analysis > Tissue isolation
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2,504 | https://bio-protocol.org/exchange/protocoldetail?id=2504&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes
Rick Conzemius
IB Ivan Barišić
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2504 Views: 8616
Edited by: Modesto Redrejo-Rodriguez
Original Research Article:
The authors used this protocol in Jan 2016
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Abstract
Diagnostic assays for pathogen identification and characterization are limited either by the number of simultaneously detectable targets, which rely on multiplexing methods, or by time constraints due to cultivation-based techniques. We recently presented a 100-plex method for human pathogen characterization to identify 75 bacterial and fungal species as well as 33 clinically relevant β-lactamases (Barišić et al., 2016). By using 16S rRNA gene sequences as barcode elements in the padlock probes, and two different fluorescence channels for species and antibiotic resistance identification, we managed to cut the number of microarray probes needed by half. Consequently, we present here the protocol of an assay with a runtime of approx. 8 h and a detection limit of 105 cfu ml-1. A total of 89% of β-lactamases and 93.7% of species were identified correctly.
Keywords: Multiplex detection Human pathogens Padlock probes Species identification Antibiotic resistance identification
Background
β-Lactamases are a class of antibiotic resistance genes which provide resistance to β-lactam antibiotics, which structurally mimic D-alanyl-D-alanine, a component of the bacterial cell wall and thereby inhibit bacterial cell wall synthesis. β-Lactamases are able to hydrolyze the central component of β-lactam antibiotics, the β-lactam ring, and render them useless (Kong et al., 2010). Today, over 1,000 β-lactamases are described and a huge potential environmental reservoir exists (Bush, 2010; Brandt et al., 2017). β-Lactamases are ancient enzymes and we classify them as class A, C, and D (serine β-lactamases) with a serine catalytic site, or as class B (metallo-β-lactamases) whose active center is zinc-dependent (Hall and Barlow, 2003 and 2004). Despite their high phylogenetic age, the serine β-lactamases probably share a common ancestor and acquired a high number of SNPs due to a permanent selection pressure. Additionally, to β-lactamases, more than 500 other antibiotic resistance genes exist (Zankari et al., 2012).
Current multiplexing methods reduce the number of simultaneously detectable targets while cultivation-dependent techniques are limited by time constraints. Given these facts and the high number of pathogens of clinical importance, new methods are needed for the fast characterization and identification of pathogens, virulence factors and antibiotic resistance genes.
The gold standard of infection diagnostics takes 2-3 days and is cultivation-dependent (Marik, 2014). Additionally, PCR methods provide results at a high sensitivity and low cost, but remain impractical due to the high number of clinically relevant targets (Mussap et al., 2007; Wellinghausen et al., 2009). The current multiplex-PCR protocols are not suitable for a high number of targets and the limitations can only be overcome by microfluidic-based assays, which run a high number of analyses in parallel.
Padlock probes are linear DNA probes, which upon annealing, circularize and are then used for rolling circle amplifications (Nilsson et al., 1994; Hardenbol et al., 2005). They allow for a higher number of multiplexing and can easily be integrated into established PCR-based assays. Recently, we presented a 100-plex method based on padlock probes for pathogen characterization (75 bacterial and fungal species) as well as 33 clinically important β-lactamases (Barišić et al., 2016). By adapting this method from our previous work (Barišić et al., 2013), we increased the sensitivity and specificity of the assay and we managed to cut down the number of microarray probes needed by half by using 16S rRNA sequences as barcode elements in the padlock probes and two different fluorescence channels for species identification and antibiotic resistance characterization. Here, we present an assay to overcome time limitations and to increase the number of detectable targets. Our assay allows for the detection of up to 105 cfu ml-1 in a total of 8 h. We were able to retrieve and correctly characterize 89% of β-lactamases and to identify 93.7% of all species.
Materials and Reagents
Filter tips 10 µl, 20 µl, 200 µl and 1,250 µl (e.g., Biozym, catalog numbers: VT0200 , VT0220 , VT0240 and VT0270 )
Safe-Lock tubes 1.5 ml (Eppendorf, catalog number: 022363204 )
Falcon 15 ml conical centrifuge tubes (Corning, catalog number: 352196 )
2 ml screw-cap tube (e.g., Roche Molecular Systems, catalog number: 03358941001 )
Ritter Riplate 384 well plate PP (Ritter, catalog number: 43001-0035 )
Glass beads, acid-washed, 150-212 μm (Sigma-Aldrich, catalog number: G1145 )
Glass beads, acid-washed, 425-600 μm (Sigma-Aldrich, catalog number: G8772 )
Vantage Silylated Aldehyde Slides (CEL Associates, catalog number: VSS-25 )
LifterSlip mSeries cover slips for microarray slides, 55 µl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 25X60IM5439001LS )
PCR tubes 0.2 ml (Eppendorf, catalog number: 0030124537 )
Millex-GV 0.22 µm syringe filter units (Merck, catalog number: SLGV033RS )
50 ml tubes (e.g., Corning, catalog number: 430829 )
Disposable syringes, e.g., Omnifix 50 ml LL (B. Braun Medical, catalog number: 8508577FN )
The multiplex PCR primers (66 in total, Table S1) targeting the β-lactamase genes were ordered from Microsynth (Balgach, Switzerland) (Note 3)
The padlock probes (66 in total, Figure 1, Table S2) targeting the β-lactamases were ordered from Integrated DNA Technologies (Coralville, IA, USA)
Figure 1. Schematic illustration of a padlock probe. The 3’ and the 5’ target recognition arms bind to the multiplex PCR products of the β-lactamase genes. During the binding process, the padlock probes are circularized and subsequently ligated. The maximum distance of the padlock probe binding region to the 3’ or 5’-ends of the PCR product should not exceed 200 base pairs. Since the padlock probes gets concatenated with the PCR product upon the ligation reaction, longer distances cause an inhibition of subsequent RCAs. The C2CA sequence is needed for the circle-to-circle amplification and comprises an AluI restriction site for monomerization of the amplification products. The barcode sequence is derived from the 16S rRNA gene. This allowed us to halve the number of microarray probes because the C2CA products and 16S rRNA gene PCR products are detected on the same microarray probe but in different fluorescence channels.
The 5’-amino-modified microarray probes (274 in total, Table S3) were ordered from Microsynth (Balgach, Switzerland) (Note 3)
Nuclease-free water for PCR application comes with the Mastermix 16S Basic kit or can be purchased separately (e.g., Fresenius Kabi, Aqua bidest. ‘Fresenius’, no catalog number)
Ultrapure water, hereafter simply referred to as water or H2O (Note 1)
The universal bacterial 16S rRNA primers 45f++ (5’-GCYTAAYACATGCAAGTCGARCG-3’) and 783R (5’-TGGACTACCAGGGTATCTAATCCT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA)
The fungal 18S rRNA (ITS region) primers ITS3 (5’-GCATCGATGAAGAACGCAGC-3’) and ITS4+ (5’-TCCT-CCGCTTATTGATATGCTTAAGT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA)
The circle-to-circle amplification (C2CA) oligonucleotides C2CA- (5’-TACTCGAGGAGCTGCATACAC-3’) and C2CA+ (5’-GTGTATGCAGCTCCTCGAGTA-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA)
The Cy5-labelled hybridization control (complimentary to ‘Bsrev’) (5’-Cy5-AAGCTCACTGGCCGTCGTTTTAAA-3’) was ordered from Microsynth (Balgach, Switzerland)
T4 DNA ligase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EL0011 )
T4 polynucleotide kinase (10 U/µl) supplied with 10x reaction buffer A (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EK0031 )
ATP solution (100 mM) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0441 )
Mastermix 16S Basic, DNA-free (Molzym, catalog number: S-040-0250 ) containing a 2.5x complete master mix, Moltaq 16S DNA polymerase and PCR-grade water (Note 2)
dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0192 )
Cy5-dCTP (1 mM solution) (GE Healthcare, catalog number: PA55021 )
VentR (exo-) DNA polymerase (New England Biolabs, catalog number: M0257S ) supplied with 10x ThermoPol reaction buffer and 100 mM MgSO4
ExpressHyb hybridization solution (Takara Bio, Clontech, catalog number: 636831 )
Ampligase thermostable DNA ligase (5 U/µl) and Ampligase 10x reaction buffer (Epicentre, catalog number: A32750 )
Bovine serum albumin (BSA), molecular biology grade (New England Biolabs, catalog number: B9000S )
phi29 DNA polymerase supplied with 10x phi29 DNA polymerase reaction buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0091 )
AluI restriction enzyme (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER0011 )
Atto532-dCTP (MoBiTec, Göttingen, Germany)
SDS solution 10% for molecular biology (AppliChem, catalog number: A0676 )
Tryptic soy broth, also referred to as CASO medium (Casein-peptone soymeal-peptone broth) (Merck, catalog number: 105459 )
PBS (10x), pH 7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 70013 )
Betaine monohydrate (Sigma-Aldrich, catalog number: B2754 )
UltraPure SSC, 20x (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15557036 )
CASO medium (see Recipes)
1x phosphate-buffered saline (PBS) (see Recipes)
2x spotting buffer (6x SSC, 3 M betaine) (see Recipes)
Equipment
Pipettes (e.g., Sartorius, catalog numbers: 728020 , 728050 , 728060 and 728070 )
Microbiological incubator shaker (e.g., IKA, model: KS 4000 i control )
Tabletop centrifuge for 1.5 ml tubes (e.g., Eppendorf, model: 5424 )
Roche MagNA Lyser Instrument (Basel, Switzerland)
Thermomixer comfort (Eppendorf, Hamburg, Germany)
Epoch Microplate spectrophotometer (Biotek, Winooski, VT, USA)
Biosan DNA/RNA UV-cleaner box (Warren, MI, USA) (recommended, see Note 4)
Thermal cycler (e.g., Thermo Fisher Scientific, Applied Biosystems, model: GeneAmpTM PCR System 2700 ) (Paisley, UK)
Heraeus Megafuge 40R with a TX-750 rotor (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MegafugeTM 40R , catalog number: 75004518; TX-750 rotor: Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003180 ) and inserts for plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003617 ) and Falcon tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003608 )
Slide humidity incubation box (e.g., LabScientific, catalog number: HIC-3 )
GeneMachines Omnigrid 100 contact arrayer (Madison, WI, USA)
International Microarray Pin Stealth 3 SMP3 (Telechem, catalog number: SMP3 )
Agilent SureScan DNA Microarray Scanner (Santa Clara, CA, USA)
Autoclave
Sartorius arium pro UV ultrapure water system (Sartorius, model: arium® pro )
Software
GenePix Pro 6.0 Software (Molecular Devices LLC, Sunnyvale, CA, USA)
Microsoft Excel or any other data analysis software
ARB software package for microarray probe design (Note 3)
Primer3 for primer design (Note 3)
Procedure
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How to cite：Conzemius, R. and Barišić, I. (2017). Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes. Bio-protocol 7(16): e2504. DOI: 10.21769/BioProtoc.2504.
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Category
Microbiology > Microbial genetics > DNA
Microbiology > Microbe-host interactions > Bacterium
Molecular Biology > DNA > Genotyping
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2,505 | https://bio-protocol.org/exchange/protocoldetail?id=2505&type=0 | # Bio-Protocol Content
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Peer-reviewed
Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans
JZ Jing Zhang
RC Rui Chen
ZY Zhenyang Yu
LX Lili Xue
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2505 Views: 36799
Edited by: Peichuan Zhang
Reviewed by: Kristin Shingler
Original Research Article:
The authors used this protocol in Apr 2016
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Abstract
Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present protocol describes the steps in analyzing the superoxide dismutase (SOD) and catalase (CAT) activities in C. elegans, which is a model organism that can be used to study effects of pharmaceutical compounds and environmental pollutants. The main steps include: (1) sample preparation; (2) total protein assay; (3) SOD activity assay; (4) CAT activity assay; and (5) medium list and formula, and also data analysis and performance notes.
Keywords: SOD CAT Total protein C. elegans Protocol
Background
Biomarkers are essential to examine biological and pathogenic processes in response to a chemical, an agent or a therapeutic intervention. Various biological processes in organisms result in reactive oxygen species (ROS) which cause oxidative stress. In response to such oxidative stress, organisms can deploy superoxide dismutase (SOD) and catalase (CAT) to scavenge ROS so as to protect the cellular homeostasis (Balaban et al., 2005). On the one hand, various chemicals (pollutants) can retard such antioxidant responses, and disturb the health of organisms including human beings. On the other hand, many pharmaceuticals aim to strengthen the antioxidant responses to improve health. Therefore, activities of SOD and CAT are very important to reflect potential effects of chemicals or/and pharmaceuticals.
Caenorhabditis elegans (C. elegans) is a model organism that has been used to study effects of pharmaceutical compounds (Dengg and van Meel, 2004; Carretero et al., 2017) and environmental pollutants (Yu et al., 2013a and 2017). Several studies have used SOD and CAT assays to indicate the antioxidant responses and potential mechanism underlying the toxic effects of test chemicals (Feng et al., 2015; Yu et al., 2012; 2016 and 2017). However, these studies simply described that the determination was carried out, by using a generic kit protocol without species-specific modifications. Therefore, the explicit protocols to perform SOD and CAT assays in C. elegans are still needed for better specific instruction.
In the present protocol, we provide a nematode protocol with experimental details to analyze SOD and CAT activities in C. elegans.
Materials and Reagents
Pipette tips (https://online-shop.eppendorf.com)
Plate (60 mm)
Centrifuge tubes, 1.5 ml (Eppendorf, catalog number: 022364111 )
Absorbent paper (KCWW, Kimberly-Clark, catalog number: 0131 )
96-well plate, with lids (Corning, Costar®, catalog number: 3599 )
Sealing tape, for 96-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 15036 )
Nematodes (wild type N2)
Note: These nematodes are treated according to each researcher’s experiments. In the present protocol, the nematodes only have difference in numbers.
BCA protein assay kits (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 or 23227 )
Protein standard (5 mg/ml)
BCA reagent A and B
SOD assay kits (Beyotime Biotechnology, catalog number: S0101 )
Reaction initiation solution
SOD detection buffer
WST-8
Enzyme solution
Reaction initiation solution (40x)
CAT assay kits (Beyotime Biotechnology, catalog number: S0051 )
Enzyme conjugate
Wash solution 40x
Substrate A and B
Stop solution
Sodium hydrate (NaOH), Analytic Reagent (Sinopharm Chemical Reagent, catalog number: 10019762 )
Sodium hypochlorite (Antiformin, NaOCl, with 6-14% active Cl), Analytic Reagent (ALADDIN, catalog number: S101636-500 ml )
Potassium phosphate dibasic (K2HPO4), Analytic Reagent (Sinopharm Chemical Reagent, catalog number: 20032118 )
Potassium phosphate monobasic (KH2PO4), Analytic Reagent (Sinopharm Chemical Reagent, catalog number: 10017618 )
Clorox solution (see Recipes)
Phosphate buffered saline/buffer (PBS), pH 7.0 (see Recipes)
Equipment
Pipettes
Microscope
Centrifuge, Eppendorf 5417R (Eppendorf, model: 5417 R , catalog number: 01396)
Pestles, Eppendorf (Eppendorf, catalog number: F0140010 )
Motor-driven tissue grinder (Beijing Baiwan Electronic Technology, catalog number: HG215-LH-A )
Ice bath, in centrifuge tube box
Microplate reader, BioTek (BioTek Instruments, model: Epoch )
Sterilized bottle, Fisherbrand (100 ml, Fisher Scientific, catalog number: FB800100 ; 250 ml, Fisher Scientific, catalog number: FB800250 ; 500 ml, Fisher Scientific, catalog number: FB800500 )
Magnetic stir bar
Magnetic stir plate
pH meter
Incubator, Yiheng (Yiheng, model: LRH-1000F )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhang, J., Chen, R., Yu, Z. and Xue, L. (2017). Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans. Bio-protocol 7(16): e2505. DOI: 10.21769/BioProtoc.2505.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Activity
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2,506 | https://bio-protocol.org/exchange/protocoldetail?id=2506&type=0 | # Bio-Protocol Content
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Peer-reviewed
Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test
Menghon Cheah
JF James W Fawcett
MA Melissa R Andrews
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2506 Views: 14577
Edited by: Xi Feng
Reviewed by: Adler R. Dillman
Original Research Article:
The authors used this protocol in Jul 2016
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Jul 2016
Abstract
The Hargreaves test is specifically designed to assess thermal pain sensation in rodents such as rats and mice. This test has been used in experiments involving pain sensitization or recovery of thermal pain response following neural injury and regeneration. We present here a step-by-step protocol highlighted with important notes to guide first-time users through the learning process. Additionally, we have also included representative data from a rat model of sensory denervation showing how the data can be analysed to obtain meaningful results. We hope that this protocol can also assist potential users in deciding whether the Hargreaves test is a suitable test for their experiment.
Keywords: Thermal pain Hargreaves Rats Mice Behavioral testing
Background
The somatosensory system is responsible for processing sensory stimuli received from the environment. These sensory stimuli include pain, touch, pressure and vibration. To study how these stimuli are processed with the ultimate goal of repairing the system in the event of injury, neuroscientists have used a plethora of animal models and behavioral tests. Some of these experiments include administration of a noxious substance into the nervous system to investigate increased pain sensitivity (Zurowski et al., 2012), inducing a central or peripheral injury to the nervous system and then observing whether particular treatments promote neural regeneration (Andrews et al., 2009; Tan et al., 2012; Cheah et al., 2016), or developing in vivo neurodegenerative models with pathology in sensory neurons (Mellone et al., 2013). Whichever the case, having a suitable test to study the behavioral response of the animals is key.
Of all the sensations, pain is perhaps one of the most studied. Transmitted via different nociceptors, pain can be further categorized into different modalities such as thermal, mechanical and chemical. Depending on the experimental goal, all of these modalities can be assessed with a specific behavioral test. We present here our approach to the Hargreaves test which is a behavioral test designed for assessing response to thermal pain in rodents such as rats and mice (Hargreaves et al., 1988). Based on our experience, the Hargreaves test is relatively straight-forward and novel users can master it in a short period of time. With the aid of this step-by-step protocol, we hope to assist potential users in deciding if the Hargreaves test is a suitable test for them and also guide them through the learning process in an efficient manner.
Materials and Reagents
Tissue paper
Note: The animals may urinate or defecate in the enclosure. Any fluid found on the framed glass panel should be cleaned immediately as this can affect the thermal conductance of the glass panel.
Disinfectant cleaning solution (e.g., 70% ethanol)
Use a disinfectant cleaning solution to remove animal odor, urine and/or feces after each session
Animals
Note: The activity level of each animal strain can affect the test tremendously as proper plantar placement of the paws is required for the test to be performed with reliable results. For whichever strain is chosen, habituation to the enclosure should be performed in the week prior to the start of experiment in order to acclimatize the animals to the setup as well as to take baseline readings.
For rats, the more docile Lewis strain may be ideal for the test because they have a tendency to move about their enclosures minimally compared to other strains, however they may fall asleep during the test resulting in delayed responsiveness. On the other hand, the more active Lister-Hooded strain may move too much, hence making the test difficult and/or potentially invalidating the results. Sprague Dawley rats have a tendency to have moderate activity level and are thus another ideal strain for this test.
For mice, the C57BL/6 strain is generally more active than other strains such as BALB/c. The more active strains may make progressing with the test difficult although with sufficient acclimatization to the testing apparatus, any strain can be used.
Sucrose/food pellets
Optional: Feeding animal a small handful (~5-10) of sucrose/food pellets may be helpful in reducing the movement and calming the animals in their enclosure.
Equipment
Hargreaves test (Ugo Basile, catalog number: 37370 or Harvard Apparatus, catalog number: 72-6692 )
A complete set of the Hargreaves test should comprise the following components (Figures 1-3):
Controller
For manipulation of test settings such as infrared intensity, display of reaction time, and power input port
Emitter/detector vessel
With a fibre optic cable connecting to the controller for infrared emission and paw movement detection
Framed glass panel
For optimal thermal conductance, the glass panel should be kept clean and protected from damage
Animal enclosure
Large: holds up to 6 rats at a time
Small: holds up to 12 mice at a time
Large platform
Supporting columns
Figure 1. A complete setup of the Hargreaves test
Figure 2. The Hargreaves test controller and emitter/detector vessel
Figure 3. Animal enclosure. The large enclosure can hold up to 6 rats, one animal in each compartment, while the small enclosure can hold up to 12 mice.
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Cheah, M., Fawcett, J. W. and Andrews, M. R. (2017). Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test. Bio-protocol 7(16): e2506. DOI: 10.21769/BioProtoc.2506.
Cheah, M., Andrews, M. R., Chew, D. J., Moloney, E. B., Verhaagen, J., Fassler, R. and Fawcett, J. W. (2016). Expression of an activated integrin promotes long-distance sensory axon regeneration in the spinal cord. J Neurosci 36(27): 7283-7297.
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Category
Neuroscience > Behavioral neuroscience > Animal model
Neuroscience > Behavioral neuroscience > Animal model
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2,507 | https://bio-protocol.org/exchange/protocoldetail?id=2507&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Exit from Pluripotency Assay of Mouse Embryonic Stem Cells
Daniel Cirera-Salinas
Constance Ciaudo
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2507 Views: 8375
Edited by: Pengpeng Li
Original Research Article:
The authors used this protocol in Feb 2017
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Abstract
A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed (Ying et al., 2003; Betschinger et al., 2013). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation defects, we adapted this ‘exit from pluripotency assay’ (Bodak et al., 2017; Cirera-Salinas et al., 2017). The protocol described here has been successfully applied to several mESC lines and is easily transposable from one laboratory to another.
Keywords: Mouse embryonic stem cells Pluripotency Commitment 2i media RNA interference Alkaline phosphatase in vitro assay
Background
For decades, scientists have tried to identify the mechanisms underlying the differentiation potential of mESCs with general (e.g., Embryoïd Body) or directed (e.g., Neuronal Precursor Cells) differentiation protocols. Recently, the 2i culture media was discovered allowing the captivation of the naïve stem cells state in vitro (Ying et al., 2008). Betschinger and colleagues took advantage of the medium and developed a new ‘exit from pluripotency’ assay allowing the identification of novel factors involved in the commitment of mESCs (Betschinger et al., 2013). Briefly, mESCs maintained in 2i media conditions, are placed for two days in permissive media. Subsequently, the 2i media is reintroduced allowing only the survival of naïve mESCs. In this assay, wild type (WT) mESCs commit to differentiation during the two days of permissive media and die after reintroduction of 2i medium. Indeed, indicating that only two days of permissive media are sufficient to break-down the pluripotency network and commit to differentiation. Unfortunately, mutant mESCs for RNA interference pathways, i.e., Dicer and Dgcr8 genes, proliferate much slower than their WT counterparts making the assessment of the exit from pluripotency in two days with the original protocol less suitable. We decided to extend the presence of the cells in the permissive media to four days and then to reintroduce the 2i media for three more days. Only cells that do not commit during the four days in permissive media conditions will be able to survive and proliferate during the final three days in 2i media. Finally, cell survival and stemness are measured with Alkaline Phosphatase (AP) staining, as in the initial protocol.
Materials and Reagents
10 cm plate (TPP Techno Plastic Products, catalog number: 93100 )
6-well plate (TPP Techno Plastic Products, catalog number: 92006 )
10 ml pipettes (Bioswisstec, catalog number: 515210 )
5 ml pipettes (Bioswisstec, catalog number: 515205 )
15 ml Falcon tube (Greiner Bio One International, catalog number: 188271 )
Glass Pasteur pipette (HUBERLAB, catalog number: 1.1127.01 )
Mouse embryonic stem cells (E14TG2a mESC line obtained from ATCC: ES-E14TG2a) (ATCC, catalog number: CRL-1821 )
Phosphate-buffered saline (PBS) 1x (Thermo Fisher Scientific, GibcoTM, catalog number: 10010015 )
Trypsin-EDTA 0.05% (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
Alkaline phosphatase Kit (Sigma-Aldrich, catalog number: 86R-1KT )
ddH2O (Sigma-Aldrich, catalog number: 99053 )
Note: This product has been discontinued.
Gelatin (Sigma-Aldrich, catalog number: G1890 )
Dulbecco’s modified Eagle medium (DMEM) high glucose (Sigma-Aldrich, catalog number: D6429 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
Leukemia inhibitory factor (LIF) 10 millions unit/ml (Merck, catalog number: ESG1107 )
2-Mercaptoethanol (βM) 50 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
Mixture of penicillin and streptomycin (PS) (Sigma-Aldrich, catalog number: P0781 )
N2B27 (Takara Bio, catalog number: Y40002 )
MAPK/ERK inhibitor 0.4 μM (PD03) (Stemolecule PD0325901) (STEMCELL Technologies, catalog number: 72184 )
GSK3β inhibitor 3 μM (Chiron) (Stemolecule CHIR99021) (STEMCELL Technologies, catalog number: 72054 )
Acetone (Merck, catalog number: 1.00014.1000 )
Formaldehyde 37% (Sigma-Aldrich, catalog number: 47608-1L-F )
0.2% gelatin solution (see Recipes)
MES medium (see Recipes)
MEF medium (see Recipes)
2i + LIF medium (see Recipes)
Fixative solution (see Recipes)
Substrate solution (see Recipes)
Equipment
HeracallTM 150i incubator (37 °C and 8% CO2) (Thermo Fischer Scientific, Thermo ScientificTM, model: HeracallTM 150i )
Centrifuge (Eppendorf, model: 5810 )
Tissue culture hood (FASTER, model: Safe FAST Premium 209, catalog number: F00024900000 )
Millipore ScepterTM 2.0 cell counter (EMD Millipore, model: ScepterTM 2.0, catalog number: PHCC00000 ) (or any cell counter)
Inverted microscope (Nikon Instruments, model: Eclipse TS100 )
Software
ImageJ software 1.48v
Adobe Photoshop CS6
GraphPad Prism 6.0a software
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Cirera-Salinas, D. and Ciaudo, C. (2017). Exit from Pluripotency Assay of Mouse Embryonic Stem Cells. Bio-protocol 7(16): e2507. DOI: 10.21769/BioProtoc.2507.
Cirera-Salinas, D., Yu, J., Bodak, M., Ngondo, R. P., Herbert, K. M. and Ciaudo C. (2017). Non-canonical function of DGCR8 controls mESCs exit from pluripotency. J Cell Biol.
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Category
Stem Cell > Pluripotent stem cell > Cell induction
Cell Biology > Cell isolation and culture > Cell growth
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2,508 | https://bio-protocol.org/exchange/protocoldetail?id=2508&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent
Edmundo Lozoya-Gloria
IC Ivette Cornejo-Corona
Hem R. Thapa
DB Daniel R. Browne
TD Timothy P. Devarenne
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2508 Views: 8639
Edited by: Maria Sinetova
Reviewed by: Igor Cesarino
Original Research Article:
The authors used this protocol in Dec 2016
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Dec 2016
Abstract
We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.
Keywords: Botryococcus braunii CellROX Green Fluorescence Hydrophobic ROS Stress Triton X-100
Background
Among the first methods developed to detect and quantify hydrogen peroxide and other organic hydroperoxides was the use of titanium (IV) ion (MacNevin and Urone, 1953). The yellow color resulted from the completion of titanium (IV) and peroxide molecules was detected by colorimetry. This method was used to detect endogenous peroxides, and to assay the catalase activity in two varieties of pear fruits for correlation with fruit ripening (Brennan and Frenkel, 1977). Another method to detect lipid hydroperoxides is based on thiobarbituric acid (TBA) and was used to measure the deterioration of foods such as milk (Sidwell et al., 1955). Although this method did not use organic solvents, steam distillation of an acidified slurry was necessary to detect hydroperoxides, and the resulting red color was quantified spectrophotometrically. The procedures described above have several disadvantages such as low sensitivity, interference with other compounds, and use of solvents or substances which may damage the living cells. A more sensitive method was developed in which the blue fluorescence of scopoletin (6-methyl-7-hydroxy-1:2-benzopyrone) disappeared after its oxidation by peroxidase enzyme (Andreae, 1955; Perschke and Broda, 1961). This method was used to detect the H2O2 production by NADPH in the microsomes from rat liver (Thurman et al., 1972). However, scopoletin is expensive, difficult to extract, and is an extremely toxic natural compound (Ojewole and Adesina, 1983a and 1983b). On the other hand, fluorescein is a dye chemically synthesized (Baeyer, 1871) and the chemical structure was elucidated (Markuszewski and Diehl, 1980). The fluorescence of both compounds, scopoletin and fluorescein, was then explained based on their similar chemical structure. So, further development of novel fluorescent dyes more stable and versatile allowed their use in very specific applications (Cathcart et al., 1983). For instance, 2,7-dichlorohydro-fluorescein diacetate (DCFH-DA) was used to study the intracellular production of active oxygen in the brown alga Fucus evanescens (Collén and Davison, 1997). The same compound DCFH-DA was also used to detect oxidative stress tolerance by abscisic acid (ABA) in the green microalga Chlamydomonas reinhardtii (Yoshida et al., 2003). Due to the wide application of these fluorescent dyes, private companies developed other compounds with different properties and each designed for specific applications. CellROX Green Reagent was designed to detect the production of ROS in living cells. So, we chose this dye to detect ROS in early times in B. braunii living cells (Life Technologies Corp., 2012). These reagents are cell-permeable and show no or very weak fluorescence in a reduced state, but their oxidation results in a strong fluorescence. In presence of ROS, the CellROX Green Reagent undergoes oxidation and produce green fluorescence followed by its binding to the DNA in the nucleus. This fact allows us to distinguish between the fluorescence resulting from ROS and the fluorescence from the chlorophyll molecule. Furthermore, this reagent can be fixed with formaldehyde and is compatible with some detergents. These characteristics of CellROX Green Reagent made it suitable to analyze ROS production in stress conditions in cells of the colonial microalga Botryococcus braunii race B (Nonomura, 1988; Banerjee et al., 2002).
Materials and Reagents
Pipette tips 200 μl (Científica Senna, catalog number: 5-20236 )
96-well microplate polypropylene (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: 267245 )
Glass microscope slide (Corning, catalog number: 2947-75X25 )
Coverslip (Corning, catalog number: 2890-22 )
Note: This product has been discontinued.
Aluminum foil (Reynolds Wrap 15 m x 30 cm)
CellROX® Green Reagent (Thermo Fischer Scientific, InvitrogenTM, catalog number: C10444 , Excitation/Emission, 485/520 nm)
Triton X-100 (Karal, catalog number: 9015 )
Methyl jasmonate (abbreviated MeJA) (Sigma-Aldrich, catalog number: 392707-5ML )
Potassium nitrate (KNO3) (Karal, catalog number: 5082 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Karal, catalog number: 6056 )
Potassium phosphate dibasic (K2HPO4) (Karal, catalog number: 5080 )
Calcium chloride dihydrate (CaCl2·2H2O) (Karal, catalog number: 2016 )
Ethylenediaminetetraacetic acid ferric-sodium salt (Fe·Na·EDTA) (Sigma-Aldrich, catalog number: E6760-100G )
Sulfuric acid (H2SO4) (Karal, catalog number: 1032 )
Boric acid (H3BO4) (Karal, catalog number: 7021 )
Manganese sulfate monohydrate (MnSO4·H2O) (Karal, catalog number: 1069 )
Zinc sulfate monohydrate (ZnSO4·7H2O) (Karal, catalog number: 4089 )
Cupric sulfate pentahydrate (CuSO4·5H2O) (Karal, catalog number: 8024 )
Sodium molybdate dihydrate (NaMoO4·2H2O) (Karal, catalog number: 4072 )
Cobalt(II) sulfate heptahydrate (CoSO4·7H2O) (Sigma-Aldrich, catalog number: 12933 )
Note: This product has been discontinued.
Sodium chloride (NaCl) (Karal, catalog number: 6052 )
Potassium chloride (KCl) (Karal, catalog number: 5087 )
Sodium bicarbonate (NaHCO3) (Karal, catalog number: 5010 )
Sodium phosphate dibasic (Na2HPO4) (Karal, catalog number: 6005 )
Modified CHU-13 media (see Recipes)
1x phosphate-buffered saline (PBS) (see Recipes)
Equipment
1.5 L flask (Corning, PYREX®, catalog number: 4980-1XL )
Micropipettes (Mettler-Toledo International, Rainin®, catalog numbers: 17014392 , 17014382 and 17011790 )
Incubator shaker (Select BioProducts, model: IncuMixTM Incubator Shaker, catalog number: SBS256 )
Centrifuge (Labnet International, model: SpectrafugeTM 16M, catalog number: C0160 )
Optical microscope (ZEISS, model: Axio Lab.A1 ) equipped with 470 nm LED module used for fluorochrome excitation and a set of 38 Endow GFP Filters (free exchange (E) EX BP 470/40, BS FT 495, EM BP 525/50) to detect the emission of the fluorochrome
Digital camera (ZEISS, model: AxioCam ICc3 Rev.3 )
pH meter (Cole-Parmer, Jenway, model: 3510 )
Autoclave Sterilmatic (Market Forge Industries, model: STM-EL )
Software
ZEN lite 2011 (ZEISS)
GraphPad Prism version 6.00 for Mac OS X, GraphPad Software, La Jolla California USA (http://www.graphpad.com)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lozoya-Gloria, E., Cornejo-Corona, I., Thapa, H. R., Browne, D. R. and Devarenne, T. P. (2017). ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent. Bio-protocol 7(16): e2508. DOI: 10.21769/BioProtoc.2508.
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Category
Plant Science > Phycology > Cell analysis
Microbiology > Microbial physiology > Stress response
Cell Biology > Cell imaging > Fluorescence
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2,509 | https://bio-protocol.org/exchange/protocoldetail?id=2509&type=0 | # Bio-Protocol Content
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Peer-reviewed
Macrophage Survival Assay Using High Content Microscopy
RE Remzi Onur Eren
NF Nicolas Fasel
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2509 Views: 6948
Original Research Article:
The authors used this protocol in Sep 2016
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Abstract
Macrophages maintain tissue homoeostasis by regulating inflammation and tissue repair mechanisms. Thus, the fate of macrophages has an impact on the state of the tissue. The aim of this protocol is to quantify macrophage survival using high content microscopy and image processing software. Here, we describe a high-content image based protocol to assess the effect of diverse stimuli in combination with pharmacological treatments on macrophage survival in a quantitative, unbiased and high-throughput manner.
Keywords: Macrophage Cell survival High-content microscope
Background
Macrophages are phagocytic innate immune cells and are the main drivers of inflammation in tissue (Medzhitov, 2008). These cells are associated with cancer together with autoimmune, autoinflammatory, infectious, neurodegenerative and metabolic diseases (Ginhoux and Jung, 2014). In this context, the role of macrophages in inflammation is well-studied, however, the impact of macrophage survival in non-infectious and infectious diseases is largely unknown. Our study showed that the activation of certain pathogen-associated receptors (PRRs) can induce macrophage survival (Eren et al., 2016). We described a molecular mechanism that demonstrated how an obligate intracellular pathogen exploits PRR-induced cell survival (Eren et al., 2016). Thus, further studies are necessary to understand the role of macrophage survival in different disease settings.
Materials and Reagents
Sterile 1.5 ml tubes (Corning, Axygen®, catalog number: MCT-175-C )
25 G-needle
50 ml syringe (B. Braun Medical, catalog number: 4617509F-02 )
Polypropylene conical 50 ml centrifuge tube (TPP Techno Plastic Products, catalog number: 91050 )
40 µM cell strainer (Corning, Falcon®, catalog number: 431750 )
90 mm Petri dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 101RTC )
96-well clear bottom cell-culture grade black imaging plates (Corning, Falcon®, catalog number: 353219 )
Sterile reagent reservoir (VWR, catalog number: 89094-664 )
10 ml serological pipette (SARSTEDT, catalog number: 86.1254.001 )
25 ml serological pipette (SARSTEDT, catalog number: 86.1685.001 )
10 μl filtered barrier tip (Biotix, Neptune®, catalog number: BT10XL )
200 μl filtered tip low retention (CLEARLINE, catalog number: 713117 )
0.22 μm syringe-filter (Carl Roth, catalog number: P668.1 )
Adhesive plate seal
6-to-9 week old specific-pathogen free C57BL/6 mice
ddH2O
Ethanol
Macrophage colony stimulating factor (M-CSF) (ImmunoTools, catalog number: 12343115 )
EDTA 0.5 M pH 8.0 solution (as described Reference 1)
Pharmacological inhibitor
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
HEPES buffer (BioConcept, catalog number: 5-31F00-H )
Penicillin-streptomycin (P/S) (BioConcept, catalog number: 4-01F00-H )
Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
Sodium hydroxide (NaOH)
Hydrochloric acid (HCl)
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 76240 )
Note: This product has been discontinued.
Cell-culture grade Ca/Mg-free Dulbecco’s PBS (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14040091 )
Saponin (Sigma-Aldrich, catalog number: 84510 )
DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
Phalloidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: A12379 )
Complete DMEM cell medium (see Recipes)
4% PFA solution (see Recipes)
5% Saponin solution (see Recipes)
Staining solution (see Recipes)
Equipment
10-11 cm long stainless steel dissecting scissors
10-11 cm long stainless steel dissecting straight forceps
Refrigerator centrifuge with 50 ml tube adapter
Cell culture incubator
Pipette controller
Pipettes
Cell counting chamber
Finnpipette® 50-300 μl 12 channel multi-pipette
Chemical hood
Luminal flow hood
Plate washer (BioTek Instruments, model: EL406 )
High content microscope (such as Molecular Devices, model: ImageXpress Micro XL )
40x Plan Apo λ 0.95 NA objective (Nikon, catalog number: MRD00405 )
pH meter
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Eren, R. and Fasel, N. (2017). Macrophage Survival Assay Using High Content Microscopy. Bio-protocol 7(16): e2509. DOI: 10.21769/BioProtoc.2509.
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Category
Immunology > Immune cell imaging > High content microscopy
Cell Biology > Cell imaging > Fluorescence
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251 | https://bio-protocol.org/exchange/protocoldetail?id=251&type=0 | # Bio-Protocol Content
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Peer-reviewed
Ribosome Fractionation in Yeast
Olesya O. Panasenko
Published: Vol 2, Iss 16, Aug 20, 2012
DOI: 10.21769/BioProtoc.251 Views: 21750
Original Research Article:
The authors used this protocol in Feb 2012
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Abstract
This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elongation by one ribosome, further ribosomes can initiate translation on the same mRNA to form polysomes. The mass of each polysomal complex is determined primarily by the number of ribosomes it contains. Hence, the population of polysomes within the cell can be size-fractionated by sucrose density gradient centrifugation on the basis of the loading of ribosomes on the mRNA. Several compounds help to maintain or to disrupt the polysomes (Figure 1).
Keywords: Ribosome Polysomes Translation Gradient centrifugation Yeast
Figure 1. Ribosome profiles after sucrose gradient centrifugation. Ribosome fractionation was performed in the presence of CHX (black), in the absence of CHX (red) or in the presence of EDTA (green). CHX stabilizes polysomes. In the absence of CHX polysomes are destroyed but 80S is still present. 80S can be completely disrupted in cell extracts by treatment with EDTA.
Materials and Reagents
Glass beads for cells breaking 0.5 mm (Bio Spec Products, catalog number: 110/9105 )
Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 ) solution 100 mg/ml prepared on ethanol
Bradford reactive (Bio-Rad Laboratories, catalog number: 500-0006 )
Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 13560400 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7625 ) 100 mM solution prepared on isopropanol
100% trichloroacetic acid (TCA) (Fluka, catalog number: 91230 )
Tubes for SW41 rotor Ti (Beckman Coulter, catalog number: 331372 )
Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 1836170 )
Lysis buffer
Ethanol
Laemmli sample buffer
Bromophenol blue
YPD media (see Recipes)
Buffer A (see Recipes)
2x buffer B2 times concentrated (see Recipes)
Sucrose gradient (see Recipes)
Foni-inert (see Recipes)
Equipment
Table Centrifuges
Glass bead beater Genie Disruptor (Scientific Industries, catalog number: SI-DD38 )
Ultracentrifuge
Rotor SW41 Ti (Beckman Coulter, catalog number: 331362 )
Density Gradient Fractionation System (ISCO, catalog number: 67-9000-177 )
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Panasenko, O. O. (2012). Ribosome Fractionation in Yeast. Bio-protocol 2(16): e251. DOI: 10.21769/BioProtoc.251.
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Category
Microbiology > Microbial cell biology > Organelle isolation
Biochemistry > Protein > Isolation and purification
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2,510 | https://bio-protocol.org/exchange/protocoldetail?id=2510&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
JI Juan Andres Imelio
NL Nicole Larrieux
AM Ariel Edgardo Mechaly
Felipe Trajtenberg
Alejandro Buschiazzo
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2510 Views: 6813
Edited by: Arsalan Daudi
Reviewed by: Yann Simon GallotQiangjun Zhou
Original Research Article:
The authors used this protocol in Dec 2016
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Abstract
We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough to allow for manual model building attaining complete atomic models. These methods contribute to tackling a major challenge in the bacterial signaling field, namely obtaining stable kinase:regulator complexes, in distinct conformational states, amenable for high-resolution crystallographic studies.
Keywords: Signaling proteins Protein phosphorylation Trapping conformational rearrangements Structure-based mutagenesis X-ray crystallography Protein engineering
Background
Structural information about bacterial signaling complexes, especially of two-component systems (TCSs), is still scarce (Casino et al., 2009; Gao and Stock, 2009). TCSs comprise a sensory histidine-kinase (HK) and a response regulator (RR) partner, present in almost all bacteria, they allow the cells to perceive the environment and to react accordingly through adaptive responses. Structural information is even more limited when it comes to TCS complexes adopting different functional states, despite the importance of such switching mechanism in signal transmission (Trajtenberg et al., 2016). We have studied the DesK-DesR pathway (de Mendoza, 2014), a TCS from Bacillus subtilis involved in regulating the cell membrane composition in adaptation to cues that reduce the bilayer’s fluidity, such as cold shock.
The protocols we have developed were aimed at overcoming major technical bottlenecks, encompassing complex purification, crystallization and X-ray structure determination. Most of these hurdles likely arise from the intrinsic flexibility and heterogeneity that characterize TCS proteins. With the purpose of trapping the DesK:DesR complex in defined signaling steps, it is useful to recall some details based on previous findings from our laboratory. The protocols have been developed to work with DesKC, a truncated DesK variant comprising the entire cytoplasmic region of DesK, without the trans-membrane sensory domain, which is catalytically competent to phosphotransfer to DesR, as well as to dephosphorylate P~DesR (Albanesi et al., 2004). As for the response regulator partner, DesR, we have chosen to use a truncated form, including the entire receiver domain (REC), competent for all DesK-mediated phosphotransfer reactions (Trajtenberg et al., 2014), but lacking the C-terminal DNA-binding domain, and thus minimizing potential inter-domain flexibility issues.
In order to trap the DesKC:DesR complex in the phosphotransfer step of the signaling pathway, we chose to use the phosphomimetic point mutant DesKC-His188Glu. This variant, when not bound to DesR, adopts a structural conformation very similar to the phosphorylated form of wild-type DesKC (Albanesi et al., 2009), hence an attractive template to mimic the phosphorylated HK just prior to the transfer reaction, also avoiding effective transfer to take place.
On the other hand, in order to trap the DesKC:DesR complex in the dephosphorylation step, previous work was instrumental by uncovering a switch mechanism of DesK, swapping between ‘active’ (kinase-on/phosphatase-off) and ‘inactive’ (phosphatase-on/kinase-off traits) states of the kinase (Albanesi et al., 2009). Briefly, the conformational transition of DesK from its kinase-active to the inhibited form, implicates the assembly of a coiled-coil structure within the central Dimerization and His-phosphotransfer (DHp) domain, a coiled-coil that is otherwise ‘broken’ when the kinase is active. The DHp, an all-helical domain, connects the trans-membrane sensor with the Catalytic ATP-binding (CA) domains, hence the identified DHp’s conformational switching plays a key role in signal transmission through long-range allosteric rearrangements. Such mechanistic insights later led to constructing a coiled-coil hyper-stabilized variant (DesKSTA) (Saita et al., 2015), harboring point-mutations at key positions (Ser150Ile, Ser153Leu and Arg157Ile) that stabilize a phosphatase-constitutive form (Saita et al., 2015). The corresponding soluble construct, with the trans-membrane domain truncated (DesKCSTAB), indeed displays a phosphatase-trapped 3D structure (Trajtenberg et al., 2016). DesKCSTAB was used to trap the DesKC:DesR complex in the dephosphorylation step, as described in this protocol.
Materials and Reagents
P2, P200 and P1000 micropipette tips, autoclaved (Gilson, catalog numbers: F161630 , F161930 and F161670 )
1.5 ml Eppendorf tubes (Eppendorf, catalog number: 022364111 )
15 and 50 ml Falcon tubes (Corning, catalog numbers: 352097 and 352098 )
Minisart 0.45 µm syringe filter (Sartorius, catalog number: 16555-K )
96-Well Clear V-Bottom 2 ml polypropylene deep well plate (Corning, catalog number: 3960 )
Minisart 0.22 µm syringe filter (Sartorius, catalog number: 16532-K )
1 L polypropylene bottles (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3140-1002 )
SnakeSkin Dialysis Tubing 3.5K MWCO, 35 mm Dry I.D., 35 feet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88244 )
Vivaspin 6 ml 10,000 MWCO centrifugal concentrator devices (Sartorius, catalog number VS0601 )
Vivaspin 20 ml 10,000 MWCO centrifugal concentrator devices (Sartorius, catalog number VS2001 )
Linbro 24-well plates (MP Biomedicals, catalog number: CPL-101 )
Cryo-loops (HAMPTON RESEARCH, catalog number: HR4-955 )
Cover slide
Escherichia coli BL21 (DE3) and TOP10F’ strains from stocks stored at -80 °C
pACYCDuet-1 (Novagen) and pQE80L (QIAGEN) plasmids
Tobacco etch virus (TEV) protease (3 mg/ml stock solution, in-house preparation)
Ultra-pure water (> 18 MΩ) filtered with 0.22 µm Express Plus filters (EMD Millipore, catalog number: SCGPT05RE )
Ethanol 95% (Industrial Uruguayan Drugstore)
Chloramphenicol (Sigma-Aldrich, catalog number: C0378 , 17 mg/ml stock solution, stored at -20 °C)
Ampicillin (Sigma-Aldrich, catalog number: A9518 , 100 mg/ml stock solution, stored at -20 °C)
Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Euromedex, catalog number: EU0008-B , 1 M stock solution, stored at -20 °C)
Lysozyme (Sigma-Aldrich, catalog number: L6876 , 100 mg/ml stock solution)
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: 229997 )
β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
Acrylamide/Bis-acrylamide 30% solution (Sigma-Aldrich, catalog number: A3574 )
DNA Ladder GeneRuler 100 bp Plus (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0321 )
Oligonucleotides for mutagenesis and PCR amplifications (IDT DNA Technologies)
Phusion High Fidelity DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F530S )
Sodium dodecyl sulfate (Sigma-Aldrich, catalog number: L5750 )
Ammonium persulfate (Sigma-Aldrich, catalog number: 248614 )
N,N,N’,N’-Tetramethylethylenediamine (Sigma-Aldrich, catalog number: T9281 )
Color Protein Ladder Prestained Broad Range (New England Biolabs, catalog number: P7712S )
Precision Plus Protein Standard All Blue (Bio-Rad Laboratories, catalog number: 1610373 )
Brilliant Blue R (Sigma-Aldrich, catalog number: B0149 )
Lithium potassium acetyl phosphate (Sigma-Aldrich, catalog number: A0262 )
Adenosine 5’-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, catalog number: A1852 )
β,γ-Methyleneadenosine 5’-triphosphate (AMP-PCP) disodium salt (Sigma-Aldrich, catalog number: M7510 )
Trizma base (Sigma-Aldrich, catalog number: T1503 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
Magnesium chloride hexahydrate (MgCl2•6H2O) (Sigma-Aldrich, catalog number: 13152 )
Polyethylene glycol (PEG) 600 (Sigma-Aldrich, catalog number: 87333 )
MES (Sigma-Aldrich, catalog number: M8250 )
Magnesium sulfate (MgSO4)
Glycerol (AppliChem, catalog number: 131339 )
Polyethylene glycol (PEG) 4000 (Sigma-Aldrich, catalog number: 95904 )
Lithium sulfate (Li2SO4) (Sigma-Aldrich, catalog number: 203653 )
Beryllium chloride (BeCl2) (Sigma-Aldrich, catalog number: 201197 )
Note: This product has been discontinued.
Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: 71519 )
Liquid nitrogen
Polyethylene glycol (PEG) 3350 (Sigma-Aldrich, catalog number: 202444 )
Tri-potassium citrate (Sigma-Aldrich, catalog number: P1722 )
Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
Tryptone plus (Sigma-Aldrich, catalog number: 61044 )
Agar (Sigma-Aldrich, catalog number: A9799 )
EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11873580001 )
Imidazole (Merck, catalog number: 104716 )
Dithiothreitol (Soltec Ventures, catalog number: M112 )
Hydrochloric acid solution (Sigma-Aldrich, catalog number: 13-1683 )
LB medium (see Recipes)
2x YT culture medium (see Recipes)
LB agar plates (see Recipes)
Lysis buffer (see Recipes)
Immobilized Metal Affinity Chromatography (IMAC) binding and washing buffer (see Recipes)
IMAC elution buffer (see Recipes)
Dialysis buffer (see Recipes)
Size Exclusion Chromatography buffer for the phosphatase complex (SEC-P buffer)
Size Exclusion Chromatography buffer for the phosphotransferase complex (SEC-PT buffer)
Equipment
Pipetman P2 single channel pipette (Gilson, catalog number: F144801 )
Pipetman P20 single channel pipette (Gilson, catalog number: F123600 )
Pipetman P200 single channel pipette (Gilson, catalog number: F123601 )
Pipetman P1000 single channel pipette (Gilson, catalog number: F123602 )
250 ml Erlenmeyer flasks (Marienfeld-Superior, catalog number: 4110207 )
5 L Erlenmeyer flasks (Marienfeld-Superior, catalog number: 4110217 )
HisTrap immobilized metal affinity chromatography (IMAC) 5 ml column with Zn-NTA resin (GE Healthcare, catalog number: 17-5248-01 . In-house preparation)
Superdex S75 26/600 (GE Healthcare, catalog number: 28989334 )
Stirring magnet (Sigma-Aldrich)
Thermomixer C (Eppendorf, model: ThermoMixer® C , catalog number: 4053-8223)
Digital Ultrasonics Sonifier S-450 cell disruptor/homogenizer (Emerson, Branson Ultrasonics, model: S-450 )
Multitron Standard incubation shaker (Infors HT)
Minispin centrifuge (Eppendorf, model: MiniSpin® , catalog number: 5452000018)
Refrigerated 5424R Centrifuge (Eppendorf, model: 5424 R , catalog number: 36-102-3795)
Refrigerated 5810R Centrifuge (Eppendorf, model: 5810 R , catalog number: 5811000010)
Refrigerated Sorvall Lynx4000 centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Lynx4000 , catalog number: 75006580)
Minipuls 3 peristaltic pump (Gilson, catalog number: F117604 )
Chromatography instrument ÄKTA purifier (GE Healthcare, model: ÄKTAxpress , catalog number: 18664501)
EPS-301 power supply (GE Healthcare, model: EPS 301, catalog number: 18-1130-01 )
Cary 50 Bio UV-visible spectrophotometer (Varian, model: Cary® 50 )
Alchemist DT (Rigaku, model: Alchemist DT )
X-ray generator MicroMax-007HF (Rigaku, model: MicroMax-007 HF )
Multilayer X-ray mirrors Varimax-HF (Rigaku, model: VariMax HF )
Image plate area detector MAR345® (marXperts, model: mar345 )
SZX16 microscope (Olympus, model: SZX16 )
EVOLT E-330 digital camera (Olympus, model: E-330 )
LG-PS2 light source (Olympus, model: LG-PS2 )
Linux Computer workstation with OS Centos 7.0
Software
Produced diffraction datasets:
Complex DesKC:DesR-REC in phosphotransferase state, low Mg2+:
http://dx.doi.org/10.15785/SBGRID/399
Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+:
http://dx.doi.org/10.15785/SBGRID/401
Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+ and BeF3-:
http://dx.doi.org/10.15785/SBGRID/408
Complex DesKC:DesR-REC in phosphatase state:
http://dx.doi.org/10.15785/SBGRID/400
Produced atomic coordinate models:
Complex DesKC:DesR-REC in phosphotransferase state, low Mg2+:
http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUJ
Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+:
http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUK
Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+ and BeF3-:
http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUL
Complex DesKC:DesR-REC in phosphatase state:
http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUN
Computational crystallography software utilized:
autoPROC (https://www.globalphasing.com/autoproc)
BUSTER (https://www.globalphasing.com/buster)
CCP4 (http://www.ccp4.ac.uk)
Phaser (http://www.phaser.cimr.cam.ac.uk)
Coot (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot)
MolProbity (http://molprobity.biochem.duke.edu)
PyMol (https://www.pymol.org)
Procedure
The use of DesKC mutants to stabilize the histidine-kinase either in its phosphatase or its phosphotransferase state
The plasmid pACYC-DesKCH188E:DesRREC encoding for the phosphomimetic DesKC variant and for the receiver domain of DesR (DesRREC), both fused to a His-tag and a TEV protease site to cleave the tag, was already available (Trajtenberg et al., 2014). This plasmid pACYC-DesKCH188E:DesRREC is thus used to co-express the following two proteins:
DesKCH188E:
MGSSHHHHHHGIHMENLYFQGRKERERLEEKLEDANERIAELVKLEERQRIARDLEDTLGQKLSLIGLKSDLARKLIYKDPEQAARELKSVQQTARTSLNEVRKIVSSMKGIRLKDELINIKQILEAADIMFIYEEEKWPENISLLNENILSMCLKEAVTNVVKHSQAKTCRVDIQQLWKEVVITVSDDGTFKGEENSFSKGHGLLGMRERLEFANGSLHIDTENGTKLTMAIPNNSK
Theoretical MW = 27.3 kDa (24.9 after TEV-cleavage)
Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.56
DesRREC:
MRGSHHHHHHGSGSENLYFQGSGSMISIFIAEDQQMLLGALGSLLNLEDDMEVVGKGTTGQDAVDFVKKRQPDVCIMDIEMPGKTGLEAAEELKDTGCKIIILTTFARPGYFQRAIKAGVKGYLLKDSPSEELANAIRSVMNGKRIYAPELMEDLYSEA
Theoretical MW = 17.4 kDa (15.2 after TEV-cleavage)
Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.40
Within the sequences listed in the previous point, His-tags are highlighted with blue fonts, in green, the TEV-recognition sites (cleaved proteins start at the final Gly, including it). Underlined in red, the phosphorylation sites: DesRREC bears the wild-type Asp, while DesKCH188E displays instead a Glu replacing the native His.
The phosphatase-stabilized variant of DesKC, DesKCSTAB, is insoluble when expressed by itself in Escherichia coli. This difficulty is solved by co-expressing DesKCSTAB with DesRREC, resulting in excellent yields of both proteins. A co-expression plasmid is generated (pACYC-DesKCSTAB:DesRREC), by sub-cloning DesKCSTAB from pHPKS/Pxyl-desKSTA (Saita et al., 2015) into pACYC-DesKCH188E:DesRREC (Trajtenberg et al., 2014) using restriction-free cloning (Unger et al., 2010), using primers STAB_F (5’-CCTGTATTTTCAGGGATCCGGTATTATAAAACTTCGCAAG-3’) and STAB_R (5’-GTCAGACACTGTAATCACAACTTCCTTCCAG-3’). Both recombinant proteins encoded in pACYC-DesKCSTAB:DesRREC include a His-tag and a TEV protease cleavage site.
pACYC-DesKCSTAB:DesRREC co-expresses the following two proteins:
DesKCSTAB:
MGSSHHHHHHGSGSENLYFQGSGIIKLRKEIERLEEKLEDANERIAELVKLEERQRIARDLHDTLGQKLSLIGLKSDLARKLIYKDPEQAARELKSVQQTARTSLNEVRKIVSSMKGIRLKDELINIKQILEAADIMFIYEEEKWPENISLLNENILSMCLKEAVTNVVKHSQAKTCRVDIQQLWKEVVITVSDDGTFKGEENSFSKGHGLLGMRERLEFANGSLHIDTENGTKLTMAIPNNSK
Theoretical MW = 27.8 kDa (25.6 after TEV-cleavage)
Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.55
DesRREC:
MRGSHHHHHHGSGSENLYFQGSGSMISIFIAEDQQMLLGALGSLLNLEDDMEVVGKGTTGQDAVDFVKKRQPDVCIMDIEMPGKTGLEAAEELKDTGCKIIILTTFARPGYFQRAIKAGVKGYLLKDSPSEELANAIRSVMNGKRIYAPELMEDLYSEA
Theoretical MW = 17.4 kDa (15.2 after TEV-cleavage)
Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.40
Note: the theoretical absorbance (280 nm) of a 1 mg/ml solution of the DesKC:DesR protein complex is approximately 0.5, which corresponds to both protein sequences together.
Within the sequences listed in the previous point, His-tags are highlighted with blue fonts, in green, the TEV-recognition sites (cleaved proteins start at the final Gly, including it). Underlined in orange, the three stabilizing substitutions within the coiled-coil motif of DesK’s DHp domain. Underlined in red, the phosphorylation sites.
Over-expression of DesKC:DesR complexes
The co-expression plasmid pACYC-DesKCSTAB:DesRREC (and similarly for pACYC-DesKCH188E:DesRREC), is transformed into 25 µl of competent Escherichia coli BL21 (DE3) cells (approximately 108 cells/ml) using heat shock for 2 min at 42 °C. A pQE80L plasmid including the DesRREC sequence (Trajtenberg et al., 2014) is co-transformed to produce a stoichiometric excess of DesRREC in the cells. 100 μl of transformed cells are spread on LB agar plates containing 100 μg/ml ampicillin and 17 μg/ml chloramphenicol, and grown overnight at 37 °C.
Between 5 and 10 single colonies are picked and inoculated in 3 ml LB culture media (to be used as pre-cultures in the next step), 100 μg/ml ampicillin and 17 μg/ml chloramphenicol, and further grown at 37 °C for 16 h with agitation (220 rpm).
Pre-cultures are inoculated 1/500 in 250 ml Erlenmeyer flasks containing 50 ml LB media, 100 μg/ml ampicillin and 17 μg/ml chloramphenicol, and further grown at 37 °C for 16 h with agitation (220 rpm).
Larger-scale cultures are launched using three 5 L Erlenmeyer flasks, each containing 1 L 2x YT culture media, 2 mM magnesium sulfate, 100 μg/ml ampicillin, 17 μg/ml chloramphenicol and a 1/100 inoculum of pre-cultured bacteria. Cultures are grown at 37 °C with agitation (220 rpm) until absorbance at wavelength 660 nm (Abs660 nm) reaches 0.7-1 (approximately 3 h). Cultures are quickly shifted to a shaker that has been pre-equilibrated at 20 °C, and further growth is continued for 30 min with agitation (220 rpm).
IPTG is added (0.5 mM final concentration) into the cultures. Induction of over-expression is achieved at 30 °C for 4 h (in the case of DesKCSTAB:DesRREC) or at 20 °C for 16 h (for DesKCH188E:DesRREC), with agitation (220 rpm) in both cases.
Cells are harvested by centrifugation at 4,600 x g for 20 min at 4 °C in 1 L polypropylene bottles.
Pellets are thoroughly resuspended in lysis buffer (see Recipes) (5 ml per g wet weight pellet) in 50 ml Falcon tubes.
Lysozyme (0.5 mg/ml final concentration) and Triton X-100 (1% vol/vol final concentration) are added to resuspended pellets, and incubated at room temperature for 30 min, until the extract is viscous due to DNA release. Extracts are then transferred to -80 °C and stored for at least 2 h.
Frozen extracts are thawed in a water bath at 37 °C. Thawed extracts are sonicated with pulses of 1 sec (set to 30% amplitude) and resting intervals of 3 sec, completing a total timespan of 4 min. This operation is repeated 3-4 times, until complete reduction of viscosity. During sonication the Falcon tube is kept refrigerated with ice. This is considered the total extract, and 40 μl are stored in electrophoresis sample buffer with β-mercaptoethanol (SBβ) for later SDS-PAGE analyses.
Total extracts are centrifuged at 12,000 x g and 4 °C for 45 min. This step separates soluble species (supernatant fraction) from insoluble ones (pellet) such as inclusion bodies, non-lysed cells, insoluble proteins, etc. The supernatants are filtered through a 0.45 μm syringe filter to a new Falcon tube, and imidazole added to 40 mM final concentration (to avoid nonspecific binding to the column during IMAC). 40 μl of supernatants are stored in SBβ for SDS-PAGE analysis. Supernatants are hereafter used as the source of soluble DesKCSTAB (or DesKCH188E) and DesRREC, for structural studies.
Purification of DesKC:DesR complexes
Zinc is used as the immobilized metal, during the first purification step by IMAC. Nickel is avoided to preclude cysteine oxidation, which previously hampered crystallization due to resulting heterogeneity in DesR samples. The IMAC column is connected to the ÄKTA purifier and pre-equilibrated with binding buffer (see Recipes) (10 column volumes or CV). Samples are injected at a flow rate of 5 ml/min, and target proteins are bound to the column through their N-terminal His-tags.
IMAC washing is achieved with 15-20 CV binding buffer, until a stable Abs280 baseline is obtained. Aliquots of the flow-through material are stored in SBβ for SDS-PAGE analysis, to monitor for potential column saturation.
IMAC elution is achieved with a linear 0-100% gradient of elution buffer (see Recipes) (30 CV), at 5 ml/min. Absorbance at wavelength 280 nm is used to monitor protein elution peaks. DesKCSTAB, DesKCH188E and DesRREC typically elute at approximately 65 mM imidazole and are collected in 96-deep-well plates. 40 μl of eluted peaks are stored in SBβ for SDS-PAGE analysis.
The elution peaks of selected proteins are pooled and incubated with TEV protease (1:40 w/w TEV/target ratio). Proteolysis is performed overnight, in dialysis bags. Dialyses are performed against 200-300 volumes of dialysis buffer (see Recipes) with gentle stirring.
Samples are recovered from the dialysis bag, and filtered with 0.45 μm syringe filters. A second IMAC zinc column is used, attached to a peristaltic pump and pre-equilibrated with binding buffer (10 CV). Samples are injected into the column at a flow rate of 5 ml/min. The flow-through is now carefully collected. The TEV protease itself includes an N-terminal His-tag, hence typically excluded from the flow-through. 20 CV of elution buffer is applied to the column to elute TEV (and potentially non-digested target protein, normally absent if the procedure worked correctly) to be monitored by SDS-PAGE.
The second IMAC flow-through sample is concentrated by ultra-filtration in Vivaspin centrifuge devices at 6,300 x g and 15 °C for 20 min to a final volume of 10 ml. This is immediately filtered through a 0.22 μm syringe filter, and injected into a size exclusion chromatography (SEC) column Superdex S75 26/600, at a flow rate of 1 ml/min. The SEC column is previously equilibrated with 2 CV SEC buffer (see Recipes).
The SEC is eluted isocratically at 1 ml/min with SEC-P buffer in the case of the DesKCSTAB:DesRREC (phosphatase) complex, or with SEC-PT buffer in the case of the DesKCH188E:DesRREC (phosphotransferase) complex. Eluted fractions are collected in 96-deep-well plates. Protein elution is monitored with 280 nm wavelength absorbance (Figures 1A and 2A). 40 μl of eluted peaks are stored in SBβ for SDS-PAGE analysis.
The calculated molecular weight (MW) of the complex is approximately 65 kDa taking into account that DesKC is a homo-dimer, and that one monomer of DesRREC could be binding to DesKC. If instead the DesKC:DesRREC ratio is 2:2, the MW is anticipated to increase to ~80 kDa. According to the calibration curves, the SEC chromatograms reveal a MW of 53.5 kDa in the case of DesKCSTAB:DesRREC, and 44.9 kDa for DesKCH188E:DesRREC. The difference with the theoretical MW figures is likely due to tertiary and quaternary structure features deviating from the ideal assumptions for globular species and their hydrodynamic radii. That these SEC peaks correspond indeed to the targeted DesKC:DesR complexes is afterwards confirmed by mass spectrometry analyses and then 3D structure determination. The final yields approximated 12 mg protein per L of cell culture (or 1-2 mg protein per g of wet weight pellet), for both complexes.
Figure 1. Size-exclusion chromatography and crystallization of the DesKCSTAB:DesRREC complex. A. Elution profile of the DesKCSTAB:DesRREC protein complex in a Superdex S75 pg 26/600 size exclusion chromatography column. The peak at 140.9 ml corresponds to the complex, according to SDS-PAGE analysis. The peak at 201.5 ml corresponds to monomeric DesRREC in excess. B. Trigonal crystal of the DesKCSTAB:DesRREC complex.
Figure 2. Size-exclusion chromatography and crystallization of the DesKCH188E:DesRREC complex. A. Elution profile of the DesKCH188E:DesRREC protein complex in a Superdex S75 pg 26/600 size exclusion chromatography column. The peak at 148.5 ml corresponds to the complex, according to SDS-PAGE analysis. The peak at 198.0 ml corresponds to monomeric DesRREC in excess. B. Monoclinic crystal of the DesKCH188E:DesRREC complex.
Selected elution peaks from SEC are pooled and concentrated with Vivaspin centrifuge devices at 6,300 x g and 15 °C for 20 min. Final concentrations of 16 mg/ml for the DesKCSTAB:DesRREC complex, and 19 mg/ml for the DesKCH188E:DesRREC complex, are typically achieved. Protein concentration is determined by UV spectrophotometry at 280 nm and calculated extinction coefficients are derived from ProtParam (ExPASy, SIB Bioinformatics Resource Portal: http://www.expasy.org/tools). Proteins are stored in 25, 50 and 100 µl aliquots at -80 °C.
Prior to crystallization, the purity and integrity of samples are checked by SDS-PAGE. Samples in SBβ are heated at 100 °C for 5 min and separated by electrophoresis in 12% polyacrylamide gels ran at 200 V.
Crystallization of DesKC:DesR complexes
DI: The DesKCSTAB:DesRREC (phosphatase) complex
The protein stock solution of pure DesKCSTAB:DesRREC complex is prepared according to a recipe where the order in the addition of the reagents is critical. First MIXA is obtained by combining 7.6 μl 100 mM AMP-PCP, 3.8 μl 1 M Tris pH 8.5 and 44.6 μl buffer SEC-P. Then 95 μl of DesKCSTAB:DesRREC complex (at ~16 mg/ml in buffer SEC-P) is added to the 56 μl of MIXA. The tube is centrifuged at 16,000 x g and 4 °C for 10 min and the supernatant used for further manipulations. In this manner ~150 µl of DesKCSTAB:DesRREC complex stock solution (at ~10 mg/ml final concentration) is obtained, containing approximately 5 mM AMP-PCP, 43.7 mM Tris pH 8.5, 462.2 mM NaCl and 9.25 mM MgCl2.
Microseeding is used to speed up the process of crystallogenesis: 2 μl are drawn from drops containing previously grown crystals, and these are crushed by vigorous pipetting with P2 micropipette tips. This is used as the source of microseeds, adding 50 μl of 30% (v/v) PEG 600, 0.1 M MES pH 6, 0.15 M MgSO4 and 5% (v/v) glycerol. This seed stock solution is diluted 1/400 into an additive solution previously prepared containing 27% (v/v) PEG 600, 0.1 M MES pH 6, 0.15 M MgSO4 and 5% (v/v) glycerol.
Hanging drop crystallizations are done at 20 °C in Linbro plates. The crystallization drops contain 0.8 μl of mother liquor (30% [w/v] PEG 4000, 0.1 M Tris-HCl pH 8.5, 0.2 M Li2SO4), 2 μl of stock protein solution, and 1.2 μl of additive solution with seeds. Three drops are set on a cover slide to seal each reservoir well. Crystals typically appear in 3-4 days, growing to suitable sizes (0.5 µm) in 10 days (Figure 1B).
Crystals are cryo-protected by slowly adding 4 μl of cryo-protection solution: 32% (w/v) PEG 4000, 0.1 M Tris-HCl pH 8, 0.2 M Li2SO4, 20 mM MgCl2, 18 mM BeF3-, 5 mM AMP-PCP and 15% glycerol. BeF3- is prepared by mixing 50 μl of a 0.9 M stock solution of NaF with 9 μl of a 1 M stock solution of BeCl2, rendering a stock solution of 152 mM BeF3-. Be is extremely toxic; all solutions containing it are handled with particular caution and wastes treated appropriately, according to safety rules. Finally, crystals are briefly soaked in 100% cryoprotection solution, fished out of the drops using cryo-loops of approximately the same size as the selected specimen, flash-frozen in liquid nitrogen and stored in cryo-vials under liquid nitrogen for further use.
DII: The DesKCH188E:DesRREC (phosphotransferase) complex
The protein stock solution of pure DesKCH188E:DesRREC complex is prepared according to a recipe where the order in the addition of the reagents is critical. First MIXA is obtained by combining 5.5 μl 100 mM AMP-PCP, 2.75 μl 1 M Tris pH 8.5, 2.2 μl 1 M MgCl2 and 43.35 μl buffer SEC-PT. Then 48 μl of DesKCH188E:DesRREC complex (at ~19 mg/ml in buffer SEC-PT) are added to 8.2 μl DesRREC (at 30 mg/ml in buffer SEC-PT) and the 53.8 μl of MIXA. The tube is centrifuged at 16,000 x g and 4 °C for 10 min and the supernatant used for further manipulations. In this manner ~110 µl of DesKCH188E:DesRREC complex stock solution (at ~8.3 mg/ml final concentration) is obtained, containing approximately 5 mM AMP-PCP, 43.1 mM Tris pH 8-8.5, 271.5 mM NaCl and 20 mM MgCl2.
Hanging drop crystallizations are done at 20 °C in Linbro plates. The crystallization drops contain 2 μl of mother liquor (18% PEG 3350, 0.3 M tri-potassium citrate) and 2 μl of stock protein solution. Two drops are set on a cover slide to seal each reservoir well. Crystals appear typically in 5-6 days, growing to suitable sizes (0.5 µm) in 15 days (Figure 2B).
Crystals are cryo-protected by quick soaking in 20% PEG 3350, 0.3 M tri-potassium citrate, 5 mM AMP-PCP, 25% glycerol, 20 or 150 mM MgCl2, and 0 or 5 mM BeF3-. Crystals are fished out of the drops using cryo-loops of approximately the same size as the selected specimen, flash-frozen in liquid nitrogen, and stored in cryo-vials under liquid nitrogen for further use.
X-ray diffraction data collection.
Single crystal X-ray diffraction experiments are carried out with an in-house copper rotating-anode source (Protein Crystallography Facility, Institut Pasteur Montevideo), or with synchrotron radiation (Soleil, France).
DesKCSTAB in complex with DesRREC (PDB Id 5IUN), crystallizes in the trigonal space group P3121. Crystals are measured in the synchrotron (Beamline Proxima I, Soleil, France), collecting 180 images with 1° oscillation range and 30 sec exposure time per image (Figure 3A). The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/400).
Figure 3. X-ray diffraction of DesKCSTAB:DesRREC crystals and resulting electron density map. A. Representative frame showing the X-ray diffraction from a single DesKCSTAB:DesRREC crystal (space group P3121). B. Zoom-in on the mid-sector of the DHp domain of the DesKCSTAB:DesRREC (phosphatase) complex, illustrated in stick representation (all atoms for the kinase, and only the Cα trace for the regulator). Oxygen atoms are colored red, nitrogens in blue, and carbons distinguished according to protein and chain: green and yellow, for the two chains in the DesKCSTAB dimer, and orange and magenta for the two bound DesRREC moieties.
DesKCH188E in complex with DesRREC with high Mg2+ and BeF3- (PDB Id 5IUL), is collected in a rotating anode X-ray generator (Protein Crystallography Facility, Institut Pasteur de Montevideo, Uruguay). 593 images are collected with 0.3° oscillation range and 10 min exposure per image (Figure 4A). The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/408).
Figure 4. X-ray diffraction of DesKCH188E:DesRREC crystals and resulting electron density map. A. Representative frame showing the X-ray diffraction from a single DesKCH188E:DesRREC crystal (space group P21). B. Zoom-in on the mid-sector of the DHp domain of the DesKCH188E:DesRREC (phosphotransferase) complex, illustrated in stick representation (all atoms for the kinase, and only the Cα trace for the regulator). Oxygen atoms are colored red, nitrogens in blue, and carbons distinguished according to protein and chain: green and yellow, for the two chains in the DesKCH188E dimer, and magenta for the single asymmetrically bound DesRREC molecule.
DesKCH188E in complex with DesRREC with high Mg2+ (PDB Id 5IUK), crystallizes in the monoclinic space group P21. Crystals are measured in the synchrotron (Beamline Proxima I, Soleil, France), collecting two different sets of 1,000 frames each. The oscillation range is 0.2° and exposure time is 0.2 sec per image. The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/401).
DesKCH188E in complex with DesRREC with low Mg2+ (PDB Id 5IUJ), crystallizes in the monoclinic space group P21. Crystals are measured in the synchrotron (Beamline Proxima II, Soleil, France), collecting 110 images with 1° oscillation range and 30 sec exposure per image. The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/399).
Data analysis
Data sets are processed using the automatic pipeline autoPROC (Vonrhein et al., 2011), which uses XDS (Kabsch, 2010) for indexing/integration, and Pointless/Aimless (Evans, 2006; Evans, 2011) for data reduction and scaling, with the following comments for each case:
DesKCSTAB in complex with DesRREC (PDB Id 5IUN): the best processing statistics are achieved by integrating all images.
DesKCH188E in complex with DesRREC with high Mg2+ (PDB Id 5IUK): the best processing strategy is to integrate and scale the first 800 frames from the first data set, merging it with frames 1-530 and 650-910 from the second set.
DesKCH188E in complex with DesRREC with high Mg2+ and BeF3- (PDB Id 5IUL): all images are eventually integrated, but successful indexing is achieved by selecting frames 10-40 and 200-240 and using the 1,000 strongest reflections.
DesKCH188E in complex with DesRREC with low Mg2+ (PDB Id 5IUJ): the best processing statistics are achieved by integrating all images.
To solve the structure of DesKCSTAB in complex with DesRREC (Figure 3B), molecular replacement is used, as previously reported (Trajtenberg et al., 2016). The search probe is a model of a DesKC:DesRREC complex generated in silico (Trajtenberg et al., 2014) by superposition of a distantly related complex from Termothoga maritima (PDB Id 3DGE) (Casino et al., 2009), and partial truncation of DesKC’s DHp domain, only keeping the invariant region (residues 190-230). It must be highlighted that the in silico modeling strategy produces hundreds of models (Trajtenberg et al., 2014). Molecular replacement is performed using Phaser (McCoy et al., 2007), searching for one copy of the in silico-generated DHp-DesRREC model of the complex, and repeating this automatically with hundreds of different initial candidates as search probes. Eventually only a handful of them are good to be placed in the asymmetric unit, giving clear signals in the rotation and the translation functions calculated with default settings. Starting with the first refinement cycles, the remaining domains and a second hemi-complex are clearly visible in the electron density maps, allowing for manual model building of the whole molecules using Coot (Emsley et al., 2010). The strategy of using in silico-generated DHp-DesRREC models as search probes is critical for molecular replacement to succeed, and the final refined model proves indeed that the selected probes were similar enough to allow for molecular replacement to succeed, providing with solutions within the radius of convergence for refinement procedures (Figure 5). Using instead crystallographic models of individual partners or domains instead is ineffective, likely due to insufficient scattering mass of small search probes and/or conformational differences between isolated vs. complexed proteins.
The structures corresponding to the phosphotransferase complex are also solved by molecular replacement with Phaser as before, using the DHp-DesRREC model as search probe, and then completing as described above, guided by the electron density maps. Once more, we refer the reader to our previous report for full details of crystallographic structure determination procedures (Trajtenberg et al., 2016).
Structure refinement is performed with Buster-TNT (Bricogne et al., 2009) using standard procedures, including non-crystallographic symmetry (NCS) restraints and Translation/Libration/Screw (TLS) descriptions in each model (documented in detail within the header of each atomic coordinates file available in the PDB).
Structures are validated throughout the refinement and towards the end, using MolProbity tools (Chen et al., 2010).
Figure 5. Structural superposition of the initial model of DesK:DesR used as Molecular Replacement probe and the final refined structure. Superposition of the Cα traces of the in silico-generated model of DesKDHp:DesRREC (orange:cyan) (Trajtenberg et al., 2014), onto the final refined crystal structure of DesKCSTAB:DesRREC (green:magenta). The entire proteins for the latter are observed in the crystal structure and could thus be manually built (indicated in grey). Of note, among the hundreds of in silico-generated models to be used for molecular replacement procedures, the few that proved useful in solving the structure, are close enough to the final structure as readily seen in this illustration, explaining their utility. Nonetheless, the actual experimental structure displays substantial changes, readily detectable in this view mostly along the DesRREC domains.
Recipes
LB medium
5 g/L NaCl
5 g/L yeast extract
15 g/L tryptone plus
2x YT culture medium
5 g/L NaCl
10 g/L yeast extract
16 g/L tryptone plus
LB agar plates
300 ml LB medium
4.5 g agar
Lysis buffer
50 mM Tris-HCl pH 8
500 mM NaCl
EDTA-free cocktail of protease inhibitors
Immobilized Metal Affinity Chromatography (IMAC) binding and washing buffer
50 mM Tris-HCl pH 8
500 mM NaCl
40 mM imidazole
10% glycerol
IMAC elution buffer
50 mM Tris-HCl pH 8
500 mM NaCl
500 mM imidazole
10% glycerol
Dialysis buffer
50 mM Tris-HCl pH 8
300 mM NaCl
0.5 mM dithiothreitol
Size Exclusion Chromatography buffer for the phosphatase complex (SEC-P buffer)
20 mM Tris-HCl pH 8
500 mM NaCl
10 mM MgCl2
Size Exclusion Chromatography buffer for the phosphotransferase complex (SEC-PT buffer)
20 mM Tris-HCl pH 8
300 mM NaCl
Acknowledgments
These protocols are adapted from previous work reported by our group (Trajtenberg et al., 2016). We are grateful to the staffs at synchrotron beamlines Proxima I and II, Soleil (France), especially William Shepard; and Daniela Albanesi for providing plasmid pHPKS/Pxyl-desKSTA. This work was supported by grants from Agencia Nacional de Investigación e Innovación (ANII), Uruguay (FCE2009_1_2679;FCE2007_219); Agence Nationale de la Recherche (ANR), France (PCV06_138918); Centro de Biología Estructural del Mercosur (www.cebem-lat.org) and Fondo para la Convergencia Estructural del MERCOSUR (COF 03/11). We are also grateful to the Institut Pasteur International Network for institutional support through the IMiZA International Joint Unit.
References
Albanesi, D., Mansilla, M. C. and de Mendoza, D. (2004). The membrane fluidity sensor DesK of Bacillus subtilis controls the signal decay of its cognate response regulator. J Bacteriol 186(9): 2655-2663.
Albanesi, D., Martin, M., Trajtenberg, F., Mansilla, M. C., Haouz, A., Alzari, P. M., de Mendoza, D. and Buschiazzo, A. (2009). Structural plasticity and catalysis regulation of a thermosensor histidine kinase. Proc Natl Acad Sci U S A 106(38): 16185-16190.
Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O. S., Vonrhein, C. and Womack, T. O. (2009). BUSTER version 2.8.0. Global Phasing.
Casino, P., Rubio, V. and Marina, A.(2009). Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction. Cell 139(2): 325-336.
Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. and Richardson, D. C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66(Pt 1): 12-21.
de Mendoza, D. (2014). Temperature sensing by membranes. Annu Rev Microbiol 68: 101-116.
Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4): 486-501.
Evans, P. (2006). Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62(Pt 1): 72-82.
Evans, P. R. (2011). An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr 67(Pt 4): 282-292.
Gao, R. and Stock, A. M. (2009). Biological insights from structures of two-component proteins. Annu Rev Microbiol 63: 133-154.
Kabsch, W. (2010). Xds. Acta Crystallogr D Biol Crystallogr 66(Pt 2): 125-132.
McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. and Read, R. J. (2007). Phaser crystallographic software. J Appl Crystallogr 40(Pt 4): 658-674.
Saita, E., Abriata, L. A., Tsai, Y. T., Trajtenberg, F., Lemmin, T., Buschiazzo, A., Dal Peraro, M., de Mendoza, D. and Albanesi, D. (2015). A coiled coil switch mediates cold sensing by the thermosensory protein DesK. Mol Microbiol 98(2): 258-271.
Trajtenberg, F., Albanesi, D., Ruetalo, N., Botti, H., Mechaly, A. E., Nieves, M., Aguilar, P. S., Cybulski, L., Larrieux, N., de Mendoza, D. and Buschiazzo, A. (2014). Allosteric activation of bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation. MBio 5(6): e02105.
Trajtenberg, F., Imelio, J. A., Machado, M. R., Larrieux, N., Marti, M. A., Obal, G., Mechaly, A. E. and Buschiazzo, A. (2016). Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife 5.
Unger, T., Jacobovitch, Y., Dantes, A., Bernheim, R. and Peleg, Y. (2010). Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172(1): 34-44.
Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T. and Bricogne, G. (2011). Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D Biol Crystallogr 67(Pt 4): 293-302.
Copyright: Imelio et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Imelio, J. A., Larrieux, N., Mechaly, A. E., Trajtenberg, F. and Buschiazzo, A. A. (2017). Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography. Bio-protocol 7(16): e2510. DOI: 10.21769/BioProtoc.2510.
Trajtenberg, F., Imelio, J. A., Machado, M. R., Larrieux, N., Marti, M. A., Obal, G., Mechaly, A. E. and Buschiazzo, A. (2016). Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife 5.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Structure
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2,511 | https://bio-protocol.org/exchange/protocoldetail?id=2511&type=0 | # Bio-Protocol Content
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Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids
Sarah D. Diermeier
David L. Spector
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2511 Views: 9628
Edited by: Ralph Bottcher
Original Research Article:
The authors used this protocol in Sep 2016
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Sep 2016
Abstract
Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids are therefore a promising model system to identify and characterize novel drivers of breast cancer that would be unlikely to be identified using 2D cell lines. Antisense oligonucleotides can be used to efficiently and specifically knockdown target genes in the cell (Bennett et al., 2017). They can be taken up freely by organoids without the need for a transfection agent, making them a convenient tool for routine lab studies and screens.
Keywords: Organoids 3D cell culture Mammary tumor Breast cancer Antisense knockdown Antisense oligonucleotides
Background
Breast cancer is the most frequent malignancy in women worldwide and the second leading cause of cancer mortality in women (Siegel et al., 2017). To improve existing treatment regimens, it is critical to identify and investigate new molecular targets that have the potential to prevent breast cancer progression. We applied RNA-seq to generate a comprehensive catalog of long non-coding RNAs (lncRNAs) that are dysregulated in primary mammary tumors compared to normal mammary epithelial cells and prioritized 30 previously uncharacterized lncRNAs as Mammary Tumor Associated RNAs (MaTARs). In order to functionally assess MaTARs as key drivers of tumor progression, we performed antisense oligonucleotide (ASO)-mediated knockdown assays of all 30 MaTARs in 3D mammary tumor organoids (Diermeier et al., 2016).
ASOs are short (20-mers), single stranded DNA molecules containing phosphorothioate-modified nucleotides as well as modifications of the 2’-ribose (5-10-5 2’-MOE gapmer) (Geary et al., 2015). Upon binding of the ASO to its complementary target, the RNA-DNA duplex stimulates degradation of the lncRNA by RNase H and thereby reduces the level of the respective transcript (Wu et al., 2004). Importantly, we found that ASO uptake in primary mammary tumor cells and organoids is efficient without the use of transfection agents, a mechanism that has been studied in detail in hepatocytes (Koller et al., 2011). ASO-mediated knockdown is particularly efficient for nuclear retained lncRNAs (Lennox and Behlke, 2016).
Organoids represent an ex vivo model of mammary gland development and model branching morphogenesis in 3D culture (Ewald, 2013; Fata et al., 2007), which is driven by two physiological processes: collective cell migration and cell proliferation. As the same processes also drive tumor invasion, the mammary organoid system can be utilized to model invasive breast cancer in vitro. Loss of branching was observed upon ASO-mediated knockdown of 20 MaTARs in organoids (Diermeier et al., 2016) as well as the lncRNA Malat1 (Arun et al., 2016), indicating that these RNAs are involved in mammary tumor cell proliferation and/or collective cell migration. Hence, we suggest that antisense-mediated knockdown in mammary tumor organoids can be used to identify and characterize novel drivers of tumor progression.
Materials and Reagents
5 cm sterile cell culture dish (e.g., Corning, Falcon®, catalog number: 353002 )
Optional: Disposable plastic Cryomold (e.g., Tissue-Tek Cryomold, Electron Microscopy Sciences, catalog number: 62534-25 )
15 ml and 50 ml centrifuge tubes (e.g., Crystalgen, catalog number: 23-2265 ; Corning, Falcon®, catalog number: 352098 )
24-well cell culture plate (Greiner Bio One International, catalog number: 662160 )
Cell culture flask, 75 cm2 (e.g., Corning, Falcon®, catalog number: 353136 )
Pasteur glass pipettes (e.g., Kimble Chase Life Science and Research Products, catalog number: 63A54 )
Sterile pipette tips (e.g., Corning)
Cell strainer 70 micron (e.g., Corning, catalog number: 431751 )
0.2 ml PCR strip tubes (e.g., Corning, Axygen®, catalog number: PCR-0208-CP-C )
96-well reaction plates (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4346907 )
Optical adhesive film (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4311971 )
Mammary tumor-bearing mouse (e.g., MMTV-PyMT (Guy et al., 1992)) with palpable tumors. Optimal tumor size is ~5-10 mm in diameter
200 Proof ethyl alcohol (e.g., UltraPure, catalog number: 200CSPTP )
Sterile water
Ice
Matrigel Growth Factor reduced Basement Membrane Matrix, phenol-red free (Corning, catalog number: 356231 )
Optional: Tissue-Tek O.C.T. compound (Electron Microscopy Sciences, catalog number: 62550-12 )
Liquid N2
Cell recovery solution (Corning, catalog number: 354253 )
1x DPBS (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
TRIzol (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596018 )
GlycoBlue (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9516 )
DNAse I, amplification grade, for cDNA synthesis (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: 18068015 )
Ethylenediaminetetraacetate acid disodium salt (EDTA)
Fetal bovine serum (FBS) (e.g., VWR, product number: 1500-500 )
Gentamicin 50 mg/ml (e.g., Lonza, catalog number: 17-528Z )
Insulin from bovine pancreas (Sigma-Aldrich, catalog number: I1882-100MG )
Trypsin (e.g., Mediatech, catalog number: 25-054-CI )
Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C5138-1G )
Advanced DMEM/F12 (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 12634010 )
Bovine serum albumin (BSA) (e.g., Sigma-Aldrich, catalog number: A2153-100G )
DNase I from bovine pancreas for organoid preparation (e.g., Sigma-Aldrich, catalog number: D4263-1VL )
Pen/Strep (e.g., Sigma-Aldrich, catalog numbers: PENNA-100MU and S6501-100G )
ITS liquid media supplement 100x (Sigma-Aldrich, catalog number: I3146-5ML )
Murine FGF-basic (PeproTech, catalog number: 450-33 )
Dimethyl sulfoxide (DMSO) (e.g., Sigma-Aldrich, catalog number: D2650-5x10ML )
Nuclease-free water (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9937 )
TaqMan Reverse Transcription Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: N8080234 )
PowerUp SYBR Green Master Mix (Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: A25743 )
Chloroform, purified (e.g., Avantor Performance Materials, MACRON, catalog number: 4432-10 )
Isopropanol, molecular biology grade (e.g., Fisher Scientific, catalog number: BP2618500 )
Collagenase solution (see Recipes)
BSA solution (see Recipes)
DNase solution (see Recipes)
Organoid medium (10 ml, sufficient for 10 wells) (see Recipes)
Freezing medium (see Recipes)
cDNA Master mix (see Recipes)
qPCR Master mix (see Recipes)
Equipment
Biological safety cabinet (e.g., NuAire)
Dissection tools (e.g., 114.3 mm scissors, Sklar Surgical Instruments, catalog number: 98-104 ; forceps, Sklar Surgical Instruments, catalog number: 97-751 , sterile scalpels, e.g., Sklar Surgical Instruments, catalog number: 06-3110 )
Cell culture incubator (e.g., Heracell i Copper CO2 incubator, Thermo Fischer Scientific, Thermo ScientificTM, model: HeracellTM 150i and 240i , catalog number: 50116050)
Shaker with temperature control (e.g., Thomas Scientific, catalog number: 1222U12)
Manufacturer: Benchmark Scientific, catalog number: H1000-M .
Centrifuge for 15 and 50 ml tubes (e.g., Eppendorf, model: 5804 )
Centrifuge for 1.5 ml reaction tubes with cooling function (e.g., Eppendorf, model: 5427 R )
Vacuum suction
Micro-pipettes (e.g., Gilson, catalog number: F167300 , catalog number for Thomas Scientific: 1222N73)
Phase-contrast microscope (e.g., Nikon)
Optional: Cryo-Safe Freeze Controller (e.g., SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F18844-0000 )
NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 , catalog number: ND-2000)
PCR machine (e.g., Applied Biosystems Proflex Thermocycler, Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4484073 )
qPCR machine (e.g., Applied Biosystems StepOne Plus Real-Time PCR system, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4376600 )
-80 °C freezer (e.g., VWR, catalog number: 10160-728 )
Water bath (e.g., PolyScience, catalog number: WB10A11B )
Optional: liquid N2 freezers (e.g., VWR, catalog number: 82017-934 )
Refrigerator (4 °C)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Diermeier, S. and Spector, D. L. (2017). Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids. Bio-protocol 7(16): e2511. DOI: 10.21769/BioProtoc.2511.
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Category
Cancer Biology > General technique > Molecular biology technique
Molecular Biology > RNA > qRT-PCR
Cell Biology > Cell isolation and culture > 3D cell culture
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2,512 | https://bio-protocol.org/exchange/protocoldetail?id=2512&type=0 | # Bio-Protocol Content
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Peer-reviewed
Establishment of a Human Cell Line Persistently Infected with Sendai Virus
CC Christopher Coakley
CP Cara Peter
SF Stephanie Fabry
Saurabh Chattopadhyay
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2512 Views: 7901
Edited by: Yannick Debing
Original Research Article:
The authors used this protocol in 14-Oct 2013
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The authors used this protocol in:
14-Oct 2013
Abstract
Interferon regulatory transcription factor 3 (IRF3) is a transcription factor that upon activation by virus infection promotes the synthesis of antiviral genes, such as the interferons (Hiscott, 2007). In addition to inducing genes, IRF3 triggers antiviral apoptosis by RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA), which is independent of its transcriptional activity. RIPA protects against lethal virus infection in cells and mice (Chattopadhyay et al., 2016). In the absence of RIPA, caused by genetic ablation, chemical mutagenesis or inhibition of the pattern recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I), Sendai virus (SeV) infection does not trigger cellular apoptosis and become persistently infected (Peters et al., 2008; Chattopadhyay et al., 2013). IRF3-expressing wild type (WT) cells (U4C) undergo SeV-induced apoptosis; however, the P2.1 cells, which are deficient in IRF3 expression are not capable of triggering viral apoptosis (Figure 1). Ectopic expression of human IRF3 restores the apoptotic activity in P2.1 cells (P2.1/IRF3, Figure 1). SeV is used as a model for studying pathogenic human viruses, which are difficult to work with or require BSL3 facility. We have previously reported that both human and mouse cells can establish SeV persistence in the absence of IRF3’s apoptotic activity (Chattopadhyay et al., 2013). Here, we outline a detailed procedure for the development of a persistently SeV-infected human cell line (Figure 2), which continuously expresses viral protein and produces low levels of infectious viral particles.
Figure 1. SeV-induced apoptosis is IRF3-dependent. HT1080-derived cell lines (U4C, P2.1 and P2.1/IRF3) were infected with Sendai virus and three days post infection culture fields were photographed, scale bar represents 50 µm.
Keywords: Sendai virus Persistence IRF3 Apoptosis P2.1
Background
IRF3 is essential for initiating antiviral defense mechanisms in host cells by way of promoting transcription of antiviral genes (Hiscott, 2007; Chattopadhyay and Sen, 2017). Upon recognition of viral dsRNA by PRRs in the cell, IRF3 becomes phosphorylated, dimerizes, and translocates to the nucleus, where it binds to the interferon-sensitive response element (ISRE), and promotes transcription of type-1 interferons, e.g., IFN-β. IRF3 is also critical for triggering apoptosis via a distinct pathway, which does not require its transcriptional activity. In a series of previous studies, we have discovered the pathway, which we named RIPA that triggers apoptosis in virus-infected cells. In RIPA, IRF3 interacts with BCL-2-Associated X protein (BAX), a pro-apoptotic factor (Chattopadhyay et al., 2010). Upon binding to BAX, IRF3 translocates to the mitochondria, and initiates a signaling cascade that ultimately promotes apoptosis (Chattopadhyay et al., 2010). In the absence of IRF3 or other components of RIPA, the cells establish viral persistence when infected with Sendai virus (SeV) (Peters et al., 2008; Chattopadhyay et al., 2013). These persistent cell lines are useful for studying the full anti-viral mechanisms of cells because the cells do not undergo apoptotic cell death. In the current protocol, we provide a detailed method to create a SeV persistent human cell line, which are defective in IRF3 expression. Viral persistence is common for many viruses, which efficiently antagonize the cell death pathways of the infected cells. An in vitro approach to study persistently infected cells will reveal ways to avoid the establishment of viral persistence. It will also be evaluated in future whether the absence of RIPA can be used as a tool to generate persistently infected cells using viruses of different lifestyles.
Materials and Reagents
Materials
Pipette tips (USA Scientific)
6 cm tissue culture plates (USA Scientific, Cyto-One, catalog number: CC7672-3359 )
Cryovials (USA Scientific, catalog number: 1412-9100 )
1.5 ml Eppendorf tubes (USA Scientific)
PVDF membrane (Bio-Rad Laboratories, catalog number: 1620177 )
Autoradiography film (Denville Scientific, catalog number: E3012 )
6-well plate
Cells
U4C cells: these cells were generated from HT1080 cells and are deficient in IFN signaling
Note: These cells are maintained in DMEM containing 10% FBS, 100 international units of penicillin, 100 µg/ml streptomycin (complete DMEM).
P2.1 cells: these cells were generated from U4C cells and are deficient in IRF3 expression
Note: These cells are maintained in DMEM containing 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin (complete DMEM).
P2.1/IRF3 cells: these cells were generated by stably expressing human IRF3 in P2.1 cells and were selected under puromycin (1 µg/ml)
Note: These cells are maintained in DMEM containing 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin and puromycin (1 µg/ml).
LLC-MK2 cells (ATCC, catalog number: CCL-7 ):
Note: These cells are maintained in Medium 199 containing 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin.
Note: U4C, P2.1 and P2.1/IRF3 cells were generated in the authors’ laboratory and were described previously. See Chattopadhyay et al., 2010 and 2016. These cell lines are available from the authors upon request.
Viruses
Sendai virus (SeV) Cantell strain (Charles River laboratories)–this strain was originally obtained from ATCC (ATCC, catalog number: VR-907 )
Reagents
Dulbecco’s modified Eagle’s medium (DMEM) (Lerner Research Institute Central Cell Services, catalog number: 11-500p )
Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
Complete EDTA-free protease inhibitor (Roche Diagnostics, catalog number: 11873580001 )
Cryoprotective medium (Lonza, catalog number: 12-132A )
Phosphate-buffered saline (PBS) (Lerner Research Institute Central Cell Services, catalog number: 123-1000p )
SDS-PAGE loading buffer (Laemelli) (Bio-Rad Laboratories, catalog number: 1610737 )
10x SDS-PAGE running buffer (AMRESCO, catalog number: 0783 )
Protein assay dye (Bio-Rad Laboratories, catalog number: 5000006 )
10x transfer buffer (AMRESCO, catalog number: 0307 )
Nonfat dry milk (Bio-Rad Laboratories, catalog number: 1706404XTU )
Tris buffered saline with Tween-20 (TBST) (AMRESCO, catalog number: K873 )
Antibodies
Sendai Virus C antibody (generated in author’s laboratory) (Chattopadhyay, 2016)
Note: Another anti-SeV antibody from Abcam, catalog number: ab33988 may be used to detect the presence of SeV.
Goat-anti-rabbit secondary antibody conjugated with horseradish peroxidase was obtained from Rockland Lab (Rockland, catalog number: 611-103-122 )
ECL plus solution (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 80196 )
Medium 199 (Thermo Fisher Scientific, GibcoTM, catalog number: 11150059 )
Agar (BD, DifcoTM, catalog number: 214050 )
Guinea pig red blood cells (Colorado Serum Company, catalog number: 30100 )
Tris buffer (pH 7.4) (Affymetrix, catalog number: 22639 )
5 M sodium chloride solution (NaCl) (Affymetrix, USB, catalog number: 75888 )
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508-10G )
Sodium fluoride (Sigma-Aldrich, catalog number: S6521 )
Note: This product has been discontinued.
β-Glycerophosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G9422 )
Sodium pyrophosphate (Sigma-Aldrich, catalog number: S6422 )
Cell lysis buffer (see Recipes)
Equipment
Micropipettes (10 μl, 200 μl, 1,000 μl) (Eppendorf)
SDS-PAGE and transfer apparatus (Bio-Rad Laboratories, model: Mini PROTEAN-II )
Note: This equipment is no longer available at manufacturer (also use the same machine for agarose gel).
Vortex (Thermo Fisher Scientific)
Tissue culture incubator (at 37 °C with 5% CO2) (Thermo Fisher Scientific)
Biosafety cabinet (Baker SterilGARD)
Table top centrifuge (Eppendorf)
Heating block (at 95 °C) (Benchmark)
Rocker (Benchmark)
Rotator (Labnet)
Refrigerator (4 °C) and freezers (-20 °C and -80 °C)
Autoradiography film processor (Kodak)
Spectrophotometer (Benchmark)
Liquid Nitrogen Tank Cryosafe CM2 (D.A.I. Scientific Equipment)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Coakley, C., Peter, C., Fabry, S. and Chattopadhyay, S. (2017). Establishment of a Human Cell Line Persistently Infected with Sendai Virus. Bio-protocol 7(16): e2512. DOI: 10.21769/BioProtoc.2512.
Download Citation in RIS Format
Category
Immunology > Host defense > Human
Microbiology > Microbe-host interactions > Virus
Molecular Biology > Protein > Anti-microbial analysis
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2,513 | https://bio-protocol.org/exchange/protocoldetail?id=2513&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells
Miguel F. Diaz
Siobahn M. Evans
Scott D. Olson
Charles S. Cox Jr
Pamela L. Wenzel
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2513 Views: 8161
Reviewed by: Meenal Sinha
Original Research Article:
The authors used this protocol in May 2017
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Original research article
The authors used this protocol in:
May 2017
Abstract
The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability of MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall shear stress (WSS), on human MSC immunomodulatory function, we have utilized microfluidics to model WSS at the luminal wall of arteries. Anti-inflammatory potency of MSCs was subsequently quantified via measurement of TNF-α production by activated murine splenocytes in co-culture assays. The TNF-α suppression assay serves as a reproducible platform for functional assessment of MSC potency and demonstrates predictive value as a surrogate assay for MSC therapeutic efficacy.
Keywords: Biomechanical force Inflammation Immunomodulation Mesenchymal stem cells Potency assay TNF-α
Background
Immunomodulatory activity of mesenchymal stem cells (MSCs) is mediated by direct cellular interactions and paracrine factors (Singer and Caplan, 2011; English, 2013). MSCs are believed to originate from pericytes that associate with endothelial cells of vasculature within the bone marrow and various tissues (Sacchetti et al., 2007; Crisan et al., 2008). This unique perivascular location positions them in close proximity to inflammatory and other soluble factors in the blood stream, poising them to monitor systemic signals. Indeed, recruitment of mural cells to the endothelium is a key event in vessel maturation, and pericytes play a critical role in vascular maintenance and integrity (Benjamin et al., 1998; Schrimpf et al., 2014). Pericytes likely monitor systemic signals by fluid outflow from arterioles and capillaries through interendothelial clefts or gaps in the basement membrane, which can expose the basolateral surface of endothelial cells outside the vessel to considerable fluid frictional force, or wall shear stress (WSS), that approximates intraluminal forces (Scallan et al., 2010). MSCs and other classes of pericytes might also view the intraluminal environment from openings between vascular endothelial cells by protrusion into the vascular lumen with cytoplasmic projections much like megakaryocytes, though more typically they ensheathe the blood vessel with branching processes (Shepro and Morel, 1993; Murphy et al., 2013). In instances of inflammation or injury, for example due to trauma to the central nervous system, pericytes have been shown to migrate away from microvessels concurrent with perivascular edema and toward injured tissue in association with blood vessel sprouting (Dore-Duffy et al., 2000; Göritz et al., 2011). Cells described as having features of MSCs have been detected circulating in human peripheral blood (Zvaifler et al., 2000), though there is some controversy surrounding evidence for MSCs in the circulation of healthy and even injured individuals (Hoogduijn et al., 2014). In those cases, disruption of endothelial-pericyte interactions could be expected to exacerbate vascular hyperpermeability which could impact migration or intravasation of MSCs (Mills et al., 2013). As MSCs are anchorage-dependent cells, a likely means of motility would include attachment to the vessel wall resulting in direct exposure to intraluminal WSS. In therapeutic applications wherein MSCs are administered intravenously, WSS would be an unavoidable stimulus during handling, infusion, and trafficking (Nitzsche et al., 2017).
We have shown that WSS typical of arterial blood flow promotes signaling through focal adhesion kinase (FAK), NF-κB, and COX2 (Diaz et al., 2017; Lee et al., 2017). Increased COX2 results in elevated prostaglandin E2 (PGE2) biosynthesis. PGE2 secreted by MSCs plays a central role in regulation of innate and adaptive immune cells. Thus, MSCs exposed to WSS more potently suppress immune cell activation in the presence of inflammatory cues (Diaz et al., 2017; Lee et al., 2017). To quantify MSC immunomodulatory activity in cells exposed to fluid flow, we co-cultured MSCs and lipopolysaccharide-activated murine splenocytes in an adaptation of the commonly used mixed lymphocyte reaction (Plumas et al., 2005). TNF-α was measured by species specific ELISA to determine cytokine production from activated murine splenocytes, thus restricting analysis to immune cell activity and enabling separate determinations of cytokine production by human MSCs. Employing this assay as a surrogate measure of MSC potency, we determined that transient exposure of MSCs to fluid shear stress improved their ability to limit activation of immune cells in the presence of inflammatory stimulus. Preconditioning of MSCs by as little as 3 h of WSS in culture was an effective means of enhancing therapeutic efficacy in treatment of a rat traumatic brain injury model. These data demonstrate that WSS enhances the immunomodulatory and neuroprotective function of MSCs. Together with complementary studies implicating PGE2 as a potency marker of MSC therapeutic efficacy (Kota et al., 2017), our studies suggest that mechanotransduction could be leveraged to improve cellular therapies available for patients with neurological injury. This co-culture assay could easily be adapted for analysis of anti-inflammatory potency of MSCs subjected to a variety of treatments, including genetically engineered MSCs.
Materials and Reagents
Falcon culture treated flask, 225 cm2 (Corning, Falcon®, catalog number: 353139 )
Falcon 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
5 ml serological pipettes (MIDSCI, catalog number: MWB-5 )
Fisherbrand premium microcentrifuge tubes, 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
IBIDI µ-Slide VI0.4 ibiTreat, sterile slide (IBIDI, catalog number: 80606 )
Fisherbrand P200 Low Retention Aerosol Barrier pipet tips (Fisher Scientific, catalog number: 02-717-165 )
Falcon Petri Dish 150 x 15 mm (Corning, Falcon®, catalog number: 351058 )
Greiner Petri Dish 35 x 10 mm (Greiner Bio One International, catalog number: 627161 )
3-Stop silicone tubing, 1.52 mm I.D. (Cole-Parmer, catalog number: SK-07624-36 )
Elbow luer connector (IBIDI, catalog number: 10802 )
Falcon round bottom polypropylene tubes (Corning, Falcon®, catalog number: 352006 )
EASYStrainer, 70 μm cell sieve, sterile (Phenix Research Products, catalog number: TCG-542070 )
Falcon 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352098 )
1 cc tuberculin syringe plunger
SHARP P1000 Precision Barrier pipet tips (Denville Scientific, catalog number: P1126 )
EASYStrainer, 40 μm cell sieve, sterile (Phenix Research Products, catalog number: TCG-542040 )
10 ml serological pipettes (MIDSCI, catalog number: MWB-10 )
Fisherbrand Borosilicate glass Pasteur pipettes (Fisher Scientific, catalog number: 13-678-20C )
Paper towel
EMD-Millipore Stericup vacuum filter unit, 500 ml size (EMD Millipore, catalog number: SCGPU05RE )
Parafilm MTM (Bemis, catalog number: PM996 )
Dow Corning silastic laboratory tubing 1.57 mm I.D. x 3.18 mm O.D. (Dow Corning, catalog number: 2415569 )
Human bone marrow (BM) MSC (Whole Bone Marrow aspirates) (AllCells, catalog number: ABM001-0 ) MSCs were isolated from whole bone marrow using a Ficoll gradient followed by plastic adherence and then cultured in MSC media (see Recipes)
Note: The MSCs used for this work were prescreened for the presence of typical MSC growth, appearance and surface marker expression and expanded for stock cyro-preservation prior to its use (Sekiya et al., 2002; Dominici et al., 2006).
Male C57BL/6 mouse (THE JACKSON LABORATORY, catalog number: 000664 ); recommended age between 2-4 months old
Hyclone Dulbecco’s phosphate buffered saline (DPBS) solution, 500 ml, calcium magnesium free (GE Healthcare, HycloneTM, catalog number: SH30028.FS )
Gibco-Tryp-LE Express enzyme, 1x, 500 ml (Thermo Fisher Scientific, GibcoTM, catalog number: 12604021 )
Gibco-trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
Atlanta Biological fetal bovine serum (FBS), embryonic stem cell qualified, 500 ml (Atlanta Biologicals, catalog number: S10250 )
Red blood cell lysing buffer hybri-max (Sigma-Aldrich, catalog number: R7767-100ML )
Lipopolysaccharide, BioXtra (Sigma-Aldrich, catalog number: L6529 )
R&D Systems Mouse TNF-alpha Quantikine ELISA kit (R&D Systems, catalog number: MTA00B )
Hyclone MEM alpha modification with glutamine and nucleosides media (GE Healthcare, HycloneTM, catalog number: SH30265.FS )
Gibco Penicillin-streptomycin, 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
MSC media (see Recipes)
Equipment
Hettich Rotofix 32A with swing bucket for 15 ml and 50 ml conical tubes (Hettich Lab Technology, model: Rotofix 32A )
Sterile Hood with vacuum suction (The Baker Company, model: SterilGARD® III Advance)
Hausser Scientific Bright-LineTM counting chamber with cover glass (Hausser Scientific, catalog number: 3110V )
P2-20 XL3000i pipettor (Denville Scientific, catalog number: P3950-20A )
Note: This product has been discontinued.
P20-200 XL3000i pipettor (Denville Scientific, catalog number: P3950-200A )
Note: This product has been discontinued.
P100-1000 XL3000i pipettor (Denville Scientific, catalog number: P3950-1000A )
Note: This product has been discontinued.
Sanyo CO2 incubator (SANYO, model: MCO-18AIC )
Ismatec REGLO peristaltic 12 roller pump (Cole-Parmer, catalog number: ISM796B )
Hettich Mikro 200R refrigerated microcentrifuge (Hettich Lab Technology, model: MIKRO 200R )
Colorimetric microplate reader (Molecular Devices, model: SpectraMax M2 )
Note: This product has been discontinued.
37 °C water bath (Fisher Scientific, model: Model 210 , catalog number: 15-462-10Q)
Note: This product has been discontinued.
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Diaz, M. F., Evans, S. M., Olson, S. D., Cox, C. S. and Wenzel, P. L. (2017). A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells. Bio-protocol 7(16): e2513. DOI: 10.21769/BioProtoc.2513.
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Category
Stem Cell > Adult stem cell > Mesenchymal stem cell
Cell Biology > Cell-based analysis > Inflammatory response
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2,514 | https://bio-protocol.org/exchange/protocoldetail?id=2514&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes
GC George Carnell
KG Keith Grehan
FF Francesca Ferrara
EM Eleonora Molesti
Nigel Temperton
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2514 Views: 14869
Edited by: Longping Victor Tse
Reviewed by: Smita NairDavid Paul
Original Research Article:
The authors used this protocol in Mar 2005
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Mar 2005
Abstract
The protocol outlined represents a cost-effective, rapid and reliable method for the generation of high-titre viral pseudotype particles with the wild-type SARS-CoV spike protein on a lentiviral vector core using the widely available transfection reagent PEI. This protocol is optimized for transfection in 6-well plates; however it can be readily scaled to different production volumes according to application. This protocol has multiple benefits including the use of readily available reagents, consistent, high pseudotype virus production Relative Luminescence Units (RLU) titres and rapid generation of novel coronavirus pseudotypes for research into strain variation, tropism and immunogenicity/sero-prevalence.
Keywords: SARS coronavirus Lentiviral pseudotype Virus neutralization
Background
The production and use of pseudotyped viral particles (PV) is widely established for many viruses, and applications in the fields of serology, surveillance and vaccine development are manifold (Temperton et al., 2015; Carnell et al., 2015). PVs have proven to be powerful tools to study the effects of viral envelope glycoprotein mutations on serological outcomes, viral tropism and immunogenicity studies especially when combined with epitope information. PVs are chimeric viral constructs in which the outer (surface) glycoprotein(s) of one virus is combined with the replication-defective viral ‘core’ of another virus. PV allow for accurate, sequence-directed, sensitive antibody neutralization assays and antiviral screening to be conducted within a low biosecurity facility and offer a safe and efficient alternative to wildtype virus use, making them exquisitely beneficial for many emerging RNA viruses of pandemic potential. Many of the published protocols require modification of the SARS spike glycoprotein and/or expensive transfection reagents (Temperton, 2009). The protocol presented here utilizes the full-length, non-codon-optimized spike protein in conjunction with the low-cost transfection reagent PEI, making this protocol widely applicable to many stakeholder laboratories. Figure 1 shows a cartoon of the lentiviral SARS-CoV PV production process directed by plasmid co-transfection.
Figure 1. Cartoon representation of the production of SARS pseudotypes. HEK293T/17 cells are transfected with three plasmids, bearing the relevant genes (Lentiviral vector, packaging construct and SARS-CoV spike expression plasmid) for the production of SARS-CoV Spike bearing lentiviral pseudotypes. This figure is modified from Carnell et al. (2015).
Materials and Reagents
MultiGuard Barrier pipette tips 1-20 and 1-200 μl (Sorenson BioScience, catalog number: 30550T )
NuncTM Cell-Culture Treated Multidishes (6-well) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
Microcentrifuge tube Safe-Lock write-on graduated with lid latch 1.5 ml
Sterile syringes (10 ml), Generic
Millex-HA 0.45 µm filters (Merck, catalog number: SLHAM33SS )
96-well white plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 136101 )
HEK 293T/17 cells (ATCC, catalog number: CRL-11268 )
Huh7 cells (Cell Signaling Technology, catalog number: 300156 )
Plasmids
Glycoprotein expression plasmid: pCAGGS-SARS-CoV spike
Lentiviral vector expressing firefly luciferase: pCSFLW
Second-generation lentiviral packaging construct plasmid: p8.91 (expresses gag, pol and rev)
Note: Information on the plasmids above can be found in Temperton et al. (2005) and Carnell et al. (2015). Plasmids available from Viral Pseudotype Unit, University of Kent. [email protected]
Dulbecco’s modified Eagle medium (DMEM) with GlutaMAX (Thermo Fisher Scientific, catalog number: 31966021 ) supplemented with 10% foetal bovine serum (FBS) (Pan-Biotech, catalog number: P40-37500 ) and 1% penicillin/streptomycin (P/S) (Pan Biotech, catalog number: P06-07100 )
Gibco Reduced Serum media Opti-MEM® (Thermo Fisher Scientific, catalog number: 31985047 )
Branched Polyethyleneimine (PEI) solution at concentration of 1 mg/ml (Sigma-Aldrich, catalog number: 408727 ).
Note: PEI is dissolved in dH2O to a concentration of 1 mg/ml and the pH is adjusted to 7 using diluted (1:3) concentrated HCl.
Phosphate-buffered saline (PBS)
Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
Positive control antibody (monoclonal/polyclonal or post-infection serum) that can neutralize the SARS pseudotype
Bright GloTM luciferase assay system (Promega, catalog number: E2650 )
Equipment
Class II biosafety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM )
Water bath or incubator
Pipettes (Gilson, model: PIPETMAN® Classic, P2 , P20 , P200 and P1000 )
Optional: BIO-RAD TC20TM Automated Cell Counter (Bio-Rad Laboratories, catalog number: 1450102EDU )
Plate centrifuge (ELMI, model: SkyLine CM-6MT )
Glomax 96 luminometer (Promega, model: GloMax® 96 )
Note: This product has been discontinued.
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Carnell, G., Grehan, K., Ferrara, F., Molesti, E. and Temperton, N. (2017). An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes. Bio-protocol 7(16): e2514. DOI: 10.21769/BioProtoc.2514.
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Category
Microbiology > Microbe-host interactions > Virus
Microbiology > Microbe-host interactions > Pseudovirus
Biochemistry > Protein > Synthesis
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2,515 | https://bio-protocol.org/exchange/protocoldetail?id=2515&type=0 | # Bio-Protocol Content
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Peer-reviewed
Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis
Srustidhar Das
SB Surinder K. Batra
SR Satyanarayana Rachagani
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2515 Views: 18045
Reviewed by: Pasquale Pellegrini
Original Research Article:
The authors used this protocol in May 2016
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Abstract
Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestinal tract and is primarily comprised of Crohn’s disease (CD) and ulcerative colitis (UC). Several murine models that include both chemical induced and genetic models have been developed that mimic some aspects of either CD or UC. These models have been instrumental in our understanding of IBD. Of the chemical induced colitis models, dextran sodium sulfate (DSS) induced colitis model is a relatively simple and very widely used model of experimental colitis.
Keywords: Dextran sodium sulfate (DSS) Colitis Inflammatory bowel disease (IBD)
Background
Inflammatory bowel disease (IBD) is a complex and multifactorial disease of unknown etiology (Sartor, 2006). However, multiple factors are considered to be critical in conferring IBD susceptibility, e.g., defects in host genetics, environmental triggers, aberrant immune response against microbial and dietary antigens (Sartor, 2006). Several murine models that address specific aspects of the disease process are available (Mizoguchi, 2012). DSS induced experimental colitis is a rapid and widely used model of intestinal inflammation (Perse and Cerar, 2012). Although the exact mechanism of DSS induced colitis is not well understood, it is widely accepted that disruption of the epithelial monolayer resulting in exposure of the underlying immune system to the intestinal contents containing microbiota and microbial products (Perse and Cerar, 2012). DSS induced colonic inflammation can be adapted as acute, chronic or relapsing model of intestinal inflammation by changing the concentration, duration and cycles of administration of DSS in drinking water. In this protocol, we provide a detailed description of procedures, important considerations when performing the protocol. We have used this protocol to address the role of Muc4 in DSS induced colitis in our previous work (Das et al., 2015).
Materials and Reagents
Sterile Eppendorf tubes (National Scientific, catalog number: CN1700-BP )
Tissue Path Macrosette processing cassettes (Fisher Scientific, catalog number: 15-182-706 )
Glass slides
8-10 week old mice generated in house or obtained from the commercial vendors
Note: Mice used in the study of Das et al., (2015) were of 129/Sv and C57BL/6J mixed background. Mice in this study were littermates and included both males and females housed under specific pathogen free (SPF) conditions.
Dextran sodium sulfate (DSS) (TdB Consultancy, catalog number: DB001 , molecular weight: 35-55 kDa)
Note: Stored in a dry place at room temperature.
Liquid nitrogen
Autoclaved drinking water
Anesthesia (Isofluorane or CO2)
10% buffered formalin (Fisher Scientific, catalog number: SF100-4 )
Ethanol
Xylene
Paraffin
mirVana miRNA isolation kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1560 )
Bioanalyzer (Agilent Technologies, Waldbronn, Germany)
DNase I (QIAGEN, catalog number: 79254 )
Oligo-dT or random hexamer primers (Thermo Fisher Scientific, InvitrogenTM, catalog number: 100023441 )
SuperScript reverse transcriptase II (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
Light Cycler 480 SYBR Green mix (Roche Molecular Systems, catalog number: 04707516001 )
Hematoxylin and eosin (H & E)
Equipment
Measuring scale/Ruler or Vernier calipers
Animal weighing balance
Feeding bottles
Tissue-Tek VIP processing machine
Dissection equipment
Mouse microisolator chambers
Pestle and mortar
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Das, S., Batra, S. K. and Rachagani, S. (2017). Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis. Bio-protocol 7(16): e2515. DOI: 10.21769/BioProtoc.2515.
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Category
Cancer Biology > Inflammation > Animal models
Cell Biology > Tissue analysis > Tissue staining
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2,516 | https://bio-protocol.org/exchange/protocoldetail?id=2516&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation and Expansion of Mesenchymal Stem Cells from Murine Adipose Tissue
Natalia V. Andreeva
Alexandra A. Dalina
Alexander V. Belyavsky
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2516 Views: 12399
Reviewed by: Yanjie Li
Original Research Article:
The authors used this protocol in Dec 2016
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Dec 2016
Abstract
Mesenchymal stem cells (MSCs) are currently intensively studied due to significant promise which they represent for successful implementations of future cell therapy clinical protocols. This in turn emphasizes importance of careful preclinical studies of MSC effects in various murine disease models. The appropriate cell preparations with reproducible biological properties are important to minimize variability of results of experimental cell therapies. We describe here a simple protocol for isolation of murine MSCs from adipose tissues and their reproducible multi-log expansion under hypoxia conditions.
Keywords: Mesenchymal stem cells Adipose tissue Oxidative stress Hypoxia Expansion
Background
MSCs were identified initially by Friedenstein as cells in bone marrow with fibroblast-like morphology, adherence to the plastic and high self-renewal capacity resulting in formation of fibroblast-like colonies in vitro (Friedenstein et al., 1976; reviewed in Phinney and Sensebé, 2013). The MSCs, due to their potential applications in medicine, are currently one of the most intensively studied adult progenitor cell types. These cells can be isolated from various organs (Murray et al., 2014), and are thought to originate from the blood vessels, either as pericytes or as vessel wall cells. In addition to their capacity to differentiate along osteogenic, adipogenic and chondrogenic lineages, MSCs possess immunomodulating properties and are thought to participate in responses to tissue damage as well as to orchestrate anti-inflammatory reactions through their ability to influence macrophage polarization (Prockop, 2013; Caplan, 2016).
Given these properties, MSCs represent significant promise for successful implementations of future relevant cell therapy clinical protocols. This in turn emphasizes importance of careful preclinical studies with MSCs in various murine disease models. The ability to prepare large numbers of appropriate cell samples with reproducible biological properties is of vital importance for minimization of variability of results during development of MSC-based experimental cell therapies. However, unlike human MSCs that possess strong anti-oxidative defenses and thus grow fairly well under atmospheric oxygen conditions, mouse MSCs are much more sensitive to oxygen stress and have a limited lifespan and expansion capacity when cultured in conventional CO2 incubators. Culturing these cells under hypoxic conditions, on the contrary, significantly extends their lifespan and allows for multi-log expansion, providing sufficient amounts of cell material with reproducible properties for repeated experiments with murine experimental disease models (Boregowda et al., 2012; Krishnappa et al., 2013). In the present paper, we describe a simple protocol for isolation of murine MSCs from adipose tissue, their reproducible expansion under hypoxia conditions, as well as long-term storage.
Materials and Reagents
6 cm cell culture dish (Greiner Bio One International, catalog number: 628160 )
Sterile pipette filter tips 20 μl (Greiner Bio One International, catalog numbers: 774288 )
Sterile pipette filter tips 200 μl (Greiner Bio One International, catalog numbers: 739288 )
Sterile pipette filter tips 1,000 μl (Greiner Bio One International, catalog numbers: 740288 )
15 ml centrifuge tube (Greiner Bio One International, catalog number: 188261 )
50 ml centrifuge tube (Greiner Bio One International, catalog number: 227261 )
1.8 ml round bottom cryogenic tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 375418 )
10 cm cell culture dish (Greiner Bio One International, catalog number: 664160 )
3.5 cm cell culture dish (Greiner Bio One International, catalog number: 627160 )
Cotton wool
Vacuum filter/storage bottle system, 0.22 µm, 500 ml (Corning, catalog number: 431097 )
0.22 μm syringe filter (Sigma-Aldrich, catalog number: Z741948)
Manufacturer: GVS, catalog number: 1214220 .
10 ml syringe (SFM Hospital Products, catalog number: 534235 )
2 ml serological pipets (Greiner Bio One International, catalog numbers: 710180 )
5 ml serological pipets (Greiner Bio One International, catalog numbers: 606180 )
10 ml serological pipets (Greiner Bio One International, catalog numbers: 607107 )
25 ml serological pipets (Greiner Bio One International, catalog numbers: 760160 )
C57BL/6 mice
Sterile distilled water
Ethanol 96% (Sigma-Aldrich, catalog number: 24105 )
Note: This product has been discontinued.
Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
Dimethyl sulfoxice (DMSO) (Sigma-Aldrich, catalog number: D2650 )
Propidium iodide (PI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: P3566 )
Cell culture media components
DMEM low glucose, powder (Thermo Fisher Scientific, GibcoTM, catalog number: 31600083 )
GlutaMax (100x) (Thermo Fisher Scientific, catalog number: 35050038 )
Penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Sodium hydrogen carbonate cell culture grade (AppliChem, catalog number: A0384 )
Amphotericin B (0.25 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C2674 )
Fetal bovine serum (GE Healthcare, HyCloneTM, catalog number: SV30160.03 )
Phosphate-buffered saline (PBS), pH 7.4 tablets (Thermo Fisher Scientific, catalog number: 18912014 )
Trypsin from porcine pancreas (Sigma-Aldrich, catalog number: T4799 )
Ethylenediaminetetraacetic acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E5134 )
DMEM low glucose medium (see Recipes)
MSC isolation medium (see Recipes)
Collagenase solution (see Recipes)
MSC growth medium (see Recipes)
1x phosphate-buffered saline (PBS) pH 7.4 (see Recipes)
Trypsin solution (see Recipes)
Equipment
Sterilized surgical tools including forceps and scissors
Pipette controller (Corning, catalog number: 4091 )
Automatic single-channel pipettes, 0.5-20, 20-200 and 100-1,000 μl, Gilson-compatible (Gilson)
Analytical balance*
Thermostated shaker (Eppendorf, New BrunswickTM, model: Innova® 4000 )
Note: This item has been discontinued. Possible substitute: Eppendorf, New BrunswickTM, model: Innova® 40.
Centrifuge 5810 R (Eppendorf, model: 5810 R , catalog number: 5811000320)
Multigas incubator (SANYO, model: MCO-19M )
Note: This item has been discontinued. Possible substitute: Panasonic Healthcare, model: MCO-170M .
Laminar flow tissue culture hood*
Inverted microscope*
Hemocytometer*
Refrigerator*
Ultra-low temperature freezer (Panasonic Healthcare, catalog number: MDF-U3386S )
Locator 6 Plus Rack and Box Systems, liquid nitrogen tank (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CY50985-70 )
Autoclave*
*Note: This item can be ordered from any qualified company.
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Andreeva, N. V., Dalina, A. and Belyavsky, A. V. (2017). Isolation and Expansion of Mesenchymal Stem Cells from Murine Adipose Tissue. Bio-protocol 7(16): e2516. DOI: 10.21769/BioProtoc.2516.
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Stem Cell > Adult stem cell > Mesenchymal stem cell
Stem Cell > Adult stem cell > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell isolation
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Hi, What was the age of sacrificed mice? and the weight of adipose tissue isolated from mice? Thanks in advance
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2,517 | https://bio-protocol.org/exchange/protocoldetail?id=2517&type=0 | # Bio-Protocol Content
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Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers
YS Yuqing Shen
PV Paolo Vignali
RW Ruoning Wang
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2517 Views: 12187
Edited by: Emilie Besnard
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in mitosis, G1, S and G2 stage in a single assay. The protocol detailed here employs detergent-based hypotonic solution to rapidly permeabilize cells and allows simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3, with specific antibody within 20 min. The protocol requires only inexpensive and commercial available reagents and also enables a rapid and complete analysis of cell cycle profile.
Keywords: Cell cycle Mitosis Hypotonic buffer Propidium iodide Histone H3
Background
Cell cycle analysis by flow cytometry is mainly based on measurement of DNA content by stain with propidium iodide (PI). The stoichiometric nature of PI ensures the accurate quantification of DNA content and allows us to reveal the distribution of cells in G1, S and G2 cell cycle stage or in sub-G1 cell death stage, the latter of which is characterized by DNA fragmentation. Most of the commonly used methods for PI-based DNA quantification require fixation using alcohol or aldehyde, which is time-consuming and labor-intensive. In addition, these methods are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Therefore, we adopted and optimized a previous established hypotonic buffer to permeabilize cells, allowing simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3 (pH3), with pH3 specific antibody (Riccardi et al., 2006; Liu et al., 2016). This method enables a rapid (within 20 min) and comprehensive analysis of cell cycle profile (G1, S, G2 and M phase).
Materials and Reagents
Falcon® 5 ml round bottom polystyrene test tube (Corning, Falcon®, catalog number: 352058 )
Dulbecco’s phosphate buffered saline, no calcium, no magnesium (DPBS) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 14200166 )
Alexa Fluor® 647 rat anti-phospho-Histone H3 (pS28) (BD, BD Bioscience, catalog number: 558217 )
Sodium citrate (Na3C6H5O7·2H2O) (Sigma-Aldrich, catalog number: 1613859 )
TritonTM X-100 (Sigma-Aldrich, catalog number: T8787 )
Propidium iodide (PI) (C27H34I2N4) (Sigma-Aldrich, catalog number: P4170 )
Hypotonic lysis/PI buffer (see Recipes)
Equipment
Centrifuge (Eppendorf, model: 5424 R )
Flow cytometer (ACEA BIO, model: NovoCyte Flow Cytometer , 488-nm laser line, for excitation)
Software
FlowJo_V10 is used for analyzing the flow cytometry data
Procedure
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How to cite：Shen, Y., Vignali, P. and Wang, R. (2017). Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers. Bio-protocol 7(16): e2517. DOI: 10.21769/BioProtoc.2517.
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Category
Cancer Biology > Cell cycle checkpoints > Cell-based assays
Cell Biology > Cell-based analysis > Flow cytometry
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2,518 | https://bio-protocol.org/exchange/protocoldetail?id=2518&type=0 | # Bio-Protocol Content
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Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells
SM Shingo Miyawaki
YO Yohei Okada
HO Hideyuki Okano
KM Kyoko Miura
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2518 Views: 14737
Edited by: Andrea Puhar
Reviewed by: Kevin Patrick O’Rourke
Original Research Article:
The authors used this protocol in May 2016
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Abstract
Pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) form teratomas when transplanted into immunodeficient mice. As teratomas contain all three germ layers (endoderm, mesoderm, ectoderm), teratoma formation assay is widely used as an index of pluripotency (Evans and Kaufman, 1981; Hentze et al., 2009; Gropp et al., 2012). On the other hand, teratoma-forming tumorigenicity also represents a major risk factor impeding potential clinical applications of pluripotent stem cells (Miura et al., 2009; Okano et al., 2013). Recently, we reported that iPSCs derived from naked mole-rat lack teratoma-forming tumorigenicity when engrafted into the testes of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice due to an ES cell-expressed Ras (ERAS) and Alternative reading frame (ARF)-dependent tumor-suppression mechanism specific to this species (Miyawaki et al., 2016). Here, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas for assessing the pluripotency and tumorigenicity.
Keywords: Pluripotent stem cells Teratoma Tumorigenicity NOD/SCID mice Testis
Background
iPSCs and ESCs are exploited for applications in cell transplantation therapy for regenerative medicine. However, these cells form tumors called teratoma containing differentiated tissues when transplanted into immune-deficient mice. Therefore, the risk of their teratoma-forming tumorigenicity limits their clinical application. Several studies have reported the methods to overcome the risk of teratoma-forming tumorigeniticy (Itakura et al., 2017; Vazquez-Martin et al., 2012). Recently, we reported that iPSCs derived from naked mole-rats lack teratoma-forming tumorigenicity when engrafted into the testes of NOD/SCID mice due to species-specific activation of tumor-suppressor ARF and a disruption mutation of the oncogene ERAS (Miyawaki et al., 2016). In this protocol, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas. This approach can minimize the immune rejection due to the presence of the testicular–blood barrier (Cheng and Mruk, 2012). In addition, this approach is advantageous because transplanted cells are easily identified around the injection site even when they do not form tumors. Thus, the technique described herein is useful for assessing the pluripotency and tumorigenicity of pluripotent stem cells.
Materials and Reagents
Falcon 15 ml conical tube (Corning, Falcon®, catalog number: 352196 )
BIO-BIK 1.5 ml centrifuge tube (Ina-optika, catalog number: CF-0150 )
26-gauge needle (Terumo Medical, catalog number: NN-2613S )
Laboratory wipe (Kimwipes) (KCWW, Kimberly-Clark, catalog number: 62011 )
0.22-μm filter unit (TPP Techno Plastic Products, catalog number: 99155 )
10 cm tissue culture dish (Corning, Falcon®, catalog number: 353046 )
Pluripotent stem cells stably expressing a fluorescent marker, such as green fluorescent protein (GFP), for detection of injected cells
Note: We used naked mole-rat (NMR) iPSCs (clones 24 and 27), mouse iPSCs (clones 20D17 and 38C2: Okita et al., 2007), and human iPSCs (clone 201B7: Takahashi et al., 2007), as described in our previous study (Miyawaki et al., 2016).
Sterile phosphate-buffered saline (PBS) (NACALAI TESQUE, catalog number: 27575-31 )
0.1% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
Fetal bovine serum (Biowest, catalog number: S1820-500 )
Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D5796 )
Penicillin/streptomycin (Wako Pure Chemical Industries, catalog number: 168-23191 )
Non-essential amino acids (NACALAI TESQUE, catalog number: 06344-56 )
β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
L-Glutamine (NACALAI TESQUE, catalog number: 04260-64 )
DMEM/F12 (Sigma-Aldrich, catalog number: D6421 )
KnockOutTM Serum Replacement (Thermo Fisher Scientific, GibcoTM, catalog number: 10828028 )
Fibroblast growth factor 2 (PeproTech, catalog number: 100-18B )
Isoflurane (Wako Pure Chemical Industries, catalog number: 099-06571 )
70% ethanol
Ampicillin (Wako Pure Chemical Industries, catalog number: 012-23303 )
Paraformaldehyde (Junsei Chemical, catalog number: 58295-1201 )
Sodium hydroxide (NACALAI TESQUE, catalog number: 31511-05 )
Paraffin
Hematoxylin and eosin (HE)
iPS medium for mouse iPSCs (see Recipe 1)
iPS medium for NMR and human iPSCs (see Recipe 2)
4% paraformaldehyde (PFA) (see Recipe 3)
Equipment
Two humidified 5% CO2 cell culture incubators: 32 °C for NMR iPSCs, 37 °C for mouse and human iPSCs (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
Centrifuge (TOMY DIGITAL BIOLOGY, model: AX-501 )
Coulter counter (Beckman Coulter, catalog number: 6605698 )
Isoflurane vaporizer, chamber, and nose cone (Shinano, catalog number: SN-487-0T )
Heating pad (Nissin, model: NHP-M30N )
Operating scissors and tweezers (see Figure 1)
25 µl Hamilton syringe (Hamilton, model: 702 N, catalog number: 80400 )
Microbalance (Shimadzu, model: ATX84 )
Icebox
Figure 1. Surgical instruments used in this protocol
Software
GraphPad Prism software (GraphPad software, model: version 6.0)
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Miyawaki, S., Okada, Y., Okano, H. and Miura, K. (2017). Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells. Bio-protocol 7(16): e2518. DOI: 10.21769/BioProtoc.2518.
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Category
Cancer Biology > Cancer stem cell > Tumor formation
Stem Cell > Pluripotent stem cell > Proliferation
Cell Biology > Tissue analysis > Macroscopic observation
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2,519 | https://bio-protocol.org/exchange/protocoldetail?id=2519&type=0 | # Bio-Protocol Content
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Quantification of Membrane Damage/Cell Death Using Evan’s Blue Staining Technique
PV Preethi Vijayaraghavareddy
VA Vanitha Adhinarayanreddy
RV Ramu S Vemanna
SS Sheshshayee Sreeman
U Udayakumar Makarla
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2519 Views: 18811
Reviewed by: Michael Enos
Original Research Article:
The authors used this protocol in 14-Oct 2013
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Abstract
Membrane damage is a hallmark of both biotic and abiotic stress responses. The membrane determines the ability of a cell to sustain altered environmental conditions and hence can be used as a biomarker to assess stress-induced cell damage or death. We present an easy, quick, cost-effective, staining and spectrophotometric method to assess membrane stability of plant cells. In this method, Evan’s blue, an azo dye, is used to assay for cell viability. More specifically, Evan’s blue dye can penetrate through ruptured or destabilized membranes and stain cells. Thus, when plant cells are subjected to stress that compromises membrane integrity, the number of cells that are permeated by Evan’s blue dye will be increased compared to control cells that are not stressed. In contrast, live, healthy cells that are capable of maintaining membrane integrity do not take up Evan’s blue dye. Cells that have taken up Evan’s blue dye will have an accumulation of a blue protoplasmic stain and these stained cells can be qualitatively documented under bright field microscopy with or without the use of a camera. Furthermore, the dye can be extracted from cells that are stained by Evan’s blue dye and can be quantified spectrophotometrically. Using this analysis, the accumulation of dye in positively-stained cells correlates with the extent of cell membrane damage and thus the amount of cells that are stained with Evan’s blue dye under various conditions can be used as an indicator of cellular stress.
Keywords: Evan’s blue Membrane damage Cell death
Background
Plants are sessile organisms that are exposed to a diverse array of stress factors. The membrane is made up of lipids and glycoproteins and act as a physical, protective barrier. The fluidity of the cell membrane is altered when the cell is exposed to stress such as heat. Oxidative stress can damage cell membranes. The reactive oxygen species (ROS) associated with oxidative stress can act on membrane lipids to decrease membrane stability. An established protocol to assess membrane stability, known as Sullivan’s method, quantifies the extent of electrolyte leakage from the membrane (Sullivan and Ross, 1979). This method is time-consuming, tedious and involves several steps. Additionally, since this method usually requires exposure of the tissue to high temperature (Initial electrolyte leakage and final electrolyte leakage after boiling at high temperature), this method cannot be used to assess the instantaneous damage to membranes in plants exposed to stress. We adapted a reliable Evan’s blue staining technique that has been used by many researchers to assess cell death or membrane damage (Smith et al., 1982; Oprisko et al., 1990; Vemanna et al., 2017) for instantly monitoring stress. Evan’s blue is an acidic, non-permitting exclusion dye which stains dead or damaged cells. The dye does not enter live cells with stable membranes (Gaff and Okong’O-Ogala, 1971). One advantage of this method is that it does not subject the tissue to high temperature. Though microscopic visualization is effective, large sample sizes make this type of analysis too time consuming (Baker and Mock, 1994). We have altered our method to analyze spectrophotometrically. Evan’s blue stain can be extracted from intact cells and analyze by spectrophotometer. Our method is highly reproducible and it can be adapted to large scale phenotyping of genotypes. The other membrane penetrating dye phenosafranin can also be used but some difficulties have been reported that it will not stain the cells without nuclei (ghost cell) and also the uptake is affected by pH (Baker and Mock, 1994).
Materials and Reagents
Eppendorf tubes
Petri plates of 10 cm diameter
96-well plates or ELISA plate (Thermo Fisher Scientific, Thermo ScientifcTM, catalog number: 442404 ) or cuvette (Sigma-Aldrich, catalog number: Z276758 )
Leaf or root tissue collected during stress period (approximately 10 discs or 250 mg)
Distilled water
Evan’s blue (Sigma-Aldrich, catalog number: E2129 )
Sodium chloride (NaCl)
Potassium nitrate (KNO3)
Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O
Ammonium dihydrogen phosphate (NH4H2PO4)
Magnesium sulfate heptahydrate (MgSO4·7H2O)
Potassium chloride (KCl)
Boric acid (H3BO3)
Manganese sulfate (MnSO4·H2O)
Zinc sulfate heptahydrate (ZnSO4·7H2O)
Copper(II) sulfate pentahydrate (CuSO4·5H2O)
Molybdic acid (H2MoO4)
Na·Fe·DTPA
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
Hydrochloric acid (HCl)
Evan’s blue staining solution (see Recipe 1)
Hoagland solution (see Recipe 2)
0.1 M CaCl2 of pH 5.6 (see Recipe 3)
1% SDS (see Recipe 4)
Equipment
Pipettes
(Optional) Pestle and mortar
Tissue lyser (Tissue lyser II) (QIAGEN, catalog number: 69982 )
Centrifuge (Thermo Fisher Scientific, model: SorvallTM ST16 , catalog number: 75004240)
Light microscope (MAGNUS ANALYTICS, model: MLX )
pH meter (Systronics, model: µController Based pH system 361, catalog number: 361 )
Spectrophotometer/ELISA plate reader (Molecular Devices, model: SpectraMax Plus 384 )
Orbital shaker (Shalom Instruments, model: SLM-GR-100 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：NV, P., PA, V., Vemanna, R. S., MS, S. and Makarla, U. (2017). Quantification of Membrane Damage/Cell Death Using Evan’s Blue Staining Technique. Bio-protocol 7(16): e2519. DOI: 10.21769/BioProtoc.2519.
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Category
Plant Science > Plant cell biology > Cell staining
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell viability > Cell death
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252 | https://bio-protocol.org/exchange/protocoldetail?id=252&type=0 | # Bio-Protocol Content
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Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs
JX Jinglin L. Xie
SS Sheena D. Singh-Babak
LC Leah E. Cowen
Published: Vol 2, Iss 20, Oct 20, 2012
DOI: 10.21769/BioProtoc.252 Views: 36983
Original Research Article:
The authors used this protocol in May 2012
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Abstract
The Minimum Inhibitory Concentration (MIC) Assay is widely used to measure the susceptibility of yeasts to antifungal agents. In serial two-fold dilutions, the lowest concentration of antifungal drug that is sufficient to inhibit fungal growth is the MIC. Typically, 50% inhibitory (MIC50) or 80% inhibitory (MIC80) values are reported. To facilitate visualization of antifungal susceptibility data, heat maps are generated whereby optical density values are represented quantitatively with colour.
Materials and Reagents
Strain to be analyzed
Overnight cultures of the strains to be analyzed and parental controls
Glucose
MOPS
Yeast extract
Bactopeptone
Yeast nitrogen base
Media
RPMI medium 1640 (Life Technologies, Gibco®, catalog number: 31800-089 ) (see Recipes)
Note: It is the standard medium recommended by the CLSI standard protocol.
YPD (see Recipes)
Note: It is a rich medium commonly used to assess susceptibility of diverse mutants.
Synthetic defined medium (see Recipes)
Note: Supplemented with required amino acids to enable growth of auxotrophic strains is commonly used for experiments requiring plasmid selection.
Equipment
96-well tissue culture plate (flat bottom with lid) (SARSTEDT AG, catalog number: 83.1835 )
Microplate reader
Multichannel pipette [optional: Multichannel electronic pipette (Rainin, model: EDP3-Plus )]
Software
Soft Max Pro or equivalent spectrophotometer software (Microplate Data Processing)
Microsoft Excel (Data Analysis)
JavaTree View (Heat Map Generation, http://jtreeview.sourceforge.net/)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Xie, L., Singh-Babak, S. D. and Cowen, L. E. (2012). Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs. Bio-protocol 2(20): e252. DOI: 10.21769/BioProtoc.252.
Singh-Babak, S. D., Babak, T., Diezmann, S., Hill, J. A., Xie, J. L., Chen, Y. L., Poutanen, S. M., Rennie, R. P., Heitman, J. and Cowen, L. E. (2012). Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog 8(5): e1002718.
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Category
Microbiology > Microbial biochemistry > Other compound
Microbiology > Antimicrobial assay > Antifungal assay
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2,520 | https://bio-protocol.org/exchange/protocoldetail?id=2520&type=0 | # Bio-Protocol Content
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Protocol for HeLa Cells Infection with Escherichia coli Strains Producing Colibactin and Quantification of the Induced DNA-damage
NB Nadège Bossuet-Greif
MB Marcy Belloy
MB Michèle Boury
EO Eric Oswald
JN Jean-Philippe Nougayrede
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2520 Views: 11587
Reviewed by: Kanika Gera
Original Research Article:
The authors used this protocol in Mar 2016
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Mar 2016
Abstract
Strains of Escherichia coli bearing the pks genomic island synthesize the genotoxin colibactin. Exposure of eukaryotic cells to E. coli producing colibactin induces DNA damages, ultimately leading to cell cycle arrest, senescence and death. Here we describe a simple method to demonstrate the genotoxicity of bacteria producing colibactin following a short infection of cultured mammalian cells with pks+ E. coli.
Keywords: Escherichia coli Enterobacteria Polyketide Colibactin Genotoxin DNA damage Cell culture Infection
Background
Colibactin is a genotoxin discovered in extra-intestinal pathogenic, commensal and probiotic strains of Escherichia coli (Nougayrede et al., 2006). Colibactin is also produced by other Enterobacteriaceae, including Klebsiella pneumonia, Enterobacter aerogenes and Citrobacter koseri (Putze et al., 2009). Colibactin is a polyketide/non-ribosomal peptide hybrid compound, synthesized by a multi-enzymatic machinery, consisting of polyketide and nonribosomal peptide synthases (PKS and NRPS), tailoring and maturation enzymes, and an efflux pump (for a review: Taieb et al., 2016). This synthesis machinery is encoded on a 52 kb genomic locus, the ‘pks’ island. Colibactin induces DNA damages in eukaryotic cells infected with pks+ bacteria. The genotoxic effect induced by colibactin requires a direct contact of live pks+ bacteria with the eukaryotic cells. Indeed, no genotoxic effect is observed with killed bacteria, or with bacterial supernatants or lysates. Thus, to demonstrate the genotoxicity of colibactin producing E. coli, cultured mammalian cells (such as HeLa cells) are infected during 4 h with live pks+ bacteria. The dose of colibactin delivered to the cells varies with the number of infecting bacteria per cell (multiplicity of infection, or MOI). At the end of the 4 h infection, bacterial growth is monitored by optical density measurement (OD600 nm). Then, the cells are washed to remove the bacteria and further incubated three days with antibiotics, to allow the development of the cytopathic phenotype associated with the DNA damage. The cellular DNA damage response results in proliferation (cell cycle) arrest, cell death and senescence (Secher et al., 2013). The microscopic observation of the cell morphology reveals the cellular response to the genotoxic insult, with reduced cell numbers and a striking giant cells phenotype (called megalocytosis) due to the cell cycle arrest and cellular senescence. The genotoxic effect can be quantified by staining the cells with methylene blue, extracting the dye and measuring the optical density at 660 nm (De Rycke et al., 1996).
Materials and Reagents
Tissue culture plate 96 wells flat bottom (P96) (Corning, Falcon®, catalog number: 353072 or equivalent)
Culture flask 250 ml, 75 cm2 (Corning, Falcon®, catalog number: 353136 or equivalent)
Paper towel
HeLa cells (ATCC, catalog number: CCL-2 )
pks+ Escherichia coli strain (stored in LB 20% glycerol at -80 °C). Strains typically used as positive controls in the authors’ laboratory are probiotic strain Nissle 1917 or the commensal mouse strain NC101
Lennox L broth base (LB medium) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12780029 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Dulbecco’s modified Eagle medium (DMEM) with 25 mM HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 42430 )
Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMax Supplement, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 , or equivalent)
Non-essential amino acids solution (NEAA) 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 11140035 )
Gentamicin solution 50 mg/ml (Sigma-Aldrich, catalog number: G1397 )
Paraformaldehyde (PFA) 20% (Electron Microscopy Sciences, catalog number: 15713 )
Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
10x PBS (Sigma-Aldrich, catalog number: D1408 )
Methylene blue (RAL DIAGNOSTICS, catalog number: 310950 )
Tris-HCl 1 M pH 8.5 (Teknova, catalog number: T1085 )
Hydrochloric acid (HCl) 1 M (Merck, catalog number: 109057 )
HeLa cell culture medium (see Recipes)
Fixation solution (see Recipes)
Methylene blue staining solution (see Recipes)
Methylene blue wash buffer (see Recipes)
Methylene blue extraction solution (see Recipes)
Equipment
Micropipette Research Plus 0.1-10 µl (Dutscher, Eppendorf, catalog number: 035602 )
Micropipette PIPETMAN G 20-200 µl (Dutscher, Gilson, catalog number: 066811 )
Multichannel micropipette 30-300 µl (Dutscher, Finnpipette, catalog number: 050667N )
Incubator for bacterial culture, with shaking (Eppendorf, New BrunswickTM, model: Innova® 42 )
CO2 incubator for cell culture (Forma Scientific)
Colorimeter to measure the absorbance at 600 nm of bacterial cultures (biochrom, model: WPA CO7500 )
Microplate reader for absorbance measurement at 600 and 660 nm (TECAN Infinite Pro)
Vortex (Dutscher, model: Vortex-Genie 2 )
Inverted microscope (Olympus, model: CKX31 )
Biological safety cabinets (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM Class II )
Chemical safety hood to handle the fixative
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bossuet-Greif, N., Belloy, M., Boury, M., Oswald, E. and Nougayrede, J. (2017). Protocol for HeLa Cells Infection with Escherichia coli Strains Producing Colibactin and Quantification of the Induced DNA-damage. Bio-protocol 7(16): e2520. DOI: 10.21769/BioProtoc.2520.
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Category
Microbiology > Microbe-host interactions > Bacterium
Cell Biology > Cell imaging > Fixed-cell imaging
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2,521 | https://bio-protocol.org/exchange/protocoldetail?id=2521&type=0 | # Bio-Protocol Content
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Peer-reviewed
An ex vivo Perifusion Method for Quantitative Determination of Neuropeptide Release from Mouse Hypothalamic Explants
Ophélia Le Thuc
Jacques Noël
Carole Rovère
Published: Vol 7, Iss 16, Aug 20, 2017
DOI: 10.21769/BioProtoc.2521 Views: 7113
Edited by: Yanjie Li
Reviewed by: Alessandro Didonna
Original Research Article:
The authors used this protocol in Dec 2016
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Dec 2016
Abstract
The hypothalamus is a primary brain area which, in mammals, regulates several physiological functions that are all related to maintaining general homeostasis, by linking the central nervous system (CNS) and the periphery. The hypothalamus itself can be considered an endocrine brain region of some sort as it hosts in its different nuclei several kinds of neuropeptide-producing and -secreting neurons. These neuropeptides have specific roles and participate in the regulation of homeostasis in general, which includes the regulation of energy metabolism, feeding behavior, water intake and body core temperature for example.
As previously mentioned, in order to exert their effects, these peptides have to be produced but also, and mostly, to be secreted. In this context, it is of great importance to be able to assess how certain conditions, diseases, or treatments can actually influence the secretion of neuropeptides, thus the function of the different neuropeptidergic circuits.
One method to assess this is the perifusion of hypothalamic explants followed by quantification of peptides within the collected fractions.
Here, we explain step-by-step how to collect fractions during ex vivo perifusion of hypothalamic explants in which one can determine quantitatively neuropeptide/neurohormone release from these viable isolated tissues. Hypothalami perifusion has two great advantages over other existing assays: (1) it allows pharmacological manipulation to dissect out signaling mechanisms underlying release of different neuropeptides/neurohormones in the hypothalamic explants and, (2) it allows simultaneous experiments with different conditions on multiple hypothalami preparations, (3) it is, to our knowledge, the only method that permits the study of neuropeptide secretion in basal conditions and under repeated stimulations with the same hypothalami explants.
Keywords: Perifusion Hypothalamus Hypothalamic explants Peptide Neuropeptide Secretion Release Neuroendocrinology Endocrinology
Background
Perifusion has been regularly used to study the pancreatic islets function. Yet, this assay is on principle adaptable to any endocrine tissue and any peptide or protein secretion.
Indeed, different perifusion systems have already been used in the past and remain a valid procedure used by different research laboratories to study the neuropeptide release from hypothalami in various conditions. For example, Callewaere and colleagues published in 2006 a study in which they analyzed the effect of the chemokine SDF-1 (stromal cell-derived factor-1) on vasotocin-induced AVP (arginine-vasopressin) release. In other studies, perifusion of hypothalamic explants has also been used to analyze SRIH (somatotropin release inhibiting hormone, a.k.a. somatostatin) release from perifused hypothalami (stimulation with an extracellular perifusion with 25 mM KCl) (Tolle et al., 2001; Zizzari et al., 2007).
Yet, currently, regarding hypothalamic explants perifusion, no standardized apparatus or protocol is available. We, ourselves, adapted a protocol from the publication by Callewaere et al., 2006.
With this method, we have recently shown ex vivo how, in mouse, the chemokine CCL2 (CC-chemokine ligand 2) is able to reduce the secretion of the orexinergic hypothalamic neuropeptide Melanin-Concentrating Hormone (MCH) and thus participate in loss of both appetite and weight in a context of high-grade inflammation (Le Thuc et al., 2016).
Materials and Reagents
15 ml and/or 50 ml centrifuge tubes (Corning, Falcon®, catalog numbers: 352096 and 352070 )
Petri dishes Ø 100 mm (Corning, catalog number: 3262 )
5 ml pipettes (Corning, Falcon®, catalog number: 357543 )
10 ml pipettes (Corning, Falcon®, catalog number: 357551 )
1.5 ml microtubes (SARSTEDT, catalog number: 72.706 )
500 ml Nalgene Filtration Units–PES FASTER membranes–0.2 µm porosity (Dutscher, catalog number: 029667 )
0.22 µm filter
8-Week old mice
Minimum Essential Medium (MEM)–no glutamine, no phenol red, no HEPES (Thermo Fischer Scientific, InvitrogenTM, catalog number: 51200046 )
2 x 10-3 M Bacitracin (from Bacillus licheniformis) (Sigma-Aldrich, catalog number: 31626 )
L-Glutamine 100x (Thermo Fischer Scientific, InvitrogenTM, catalog number: 25030024 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
Protease Inhibitor cocktail (CompleteTM, EDTA-free Protease Inhibitor Cocktail) (Roche Diagnostics)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
Ultrapure water
ELISA (Phoenix Pharmaceuticals, catalog number: EK-070-47 )
Hypothalamic explant perifusion medium (see Recipes)
1 M KCl (see Recipes)
Stimulation medium with 60 mM KCl (Iso-osmotic 60 mM KCl, 50 ml) (see Recipes)
Equipment
Racks for microtubes and 15 ml/50 ml conical tubes (Dutscher)
P10, P200, P1000 pipetmen (Gilson)
-20 °C and -80 °C freezer (Sanyo, VWR)
Hole puncher (Maped–any regular hole punch from any stationary brand should do)
Carbogen (5% CO2/95% O2) (Linde France, UN 3156)
Standard Pattern Scissors, Large Loops Sharp/Blunt 14.5 cm (Fine Science Tools, catalog number: 14101-14 )
Extra Thin Iris Scissors, 10.5 cm (Fine Science Tools, catalog number: 14088-10 )
Standard Pattern Forceps with serrated tip (Fine Science Tools, catalog number: 11000-13 )
Dumont #7 forceps with curved tip (Fine Science Tools, catalog numbers: 11271-30 and 11272-30 )
2 Thermostatic baths (such as JULABO, model: CORIO CD-B27 )
Perifusion chambers (BIOREP TECHNOLOGIES, catalog number: PERI-CHAMBER )
Perifusion chamber filters (BIOREP TECHNOLOGIES, catalog number: PERI-FILTER )
Perifusion tubing set (BIOREP TECHNOLOGIES, catalog number: PERI-TUBSET )
Peristaltic pump (high precision multichannel pump, Ismatec)
Osmometer (Löser Messtechnik, model: Micro-Osmometer Type 6 )
Software
GraphPad Prism (GraphPad software) or any software for statistical data analysis
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Le Thuc, O., Noël, J. and Rovère, C. (2017). An ex vivo Perifusion Method for Quantitative Determination of Neuropeptide Release from Mouse Hypothalamic Explants. Bio-protocol 7(16): e2521. DOI: 10.21769/BioProtoc.2521.
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Category
Neuroscience > Neuroanatomy and circuitry > Brain nerve
Cell Biology > Tissue analysis > Physiology
Biochemistry > Other compound > Peptide
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2,522 | https://bio-protocol.org/exchange/protocoldetail?id=2522&type=0 | # Bio-Protocol Content
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Peer-reviewed
Using CRISPR-ERA Webserver for sgRNA Design
HL Honglei Liu
XW Xiaowo Wang
Lei S. Qi
Published: Vol 7, Iss 17, Sep 5, 2017
DOI: 10.21769/BioProtoc.2522 Views: 11816
Original Research Article:
The authors used this protocol in Nov 2015
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Abstract
The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/) is one of the state-of-the-art designer webserver tools, which has been developed both for gene editing and gene regulation sgRNA design. This protocol discusses how to design sgRNA sequences and genome-wide sgRNA library using CRISPR-ERA.
Keywords: sgRNA design CRISPR-Cas9 system sgRNA library Gene editing Gene regulation
Background
Genome engineering is essential to the study of biology, which attracted several new technological breakthroughs (Doudna and Charpentier, 2014). CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) technology has proven to have great efficiency and generalizability both in gene editing and gene regulation (Qi et al., 2013; La Russa and Qi, 2015). CRISPR-Cas9 system consists of Cas9 endonuclease and a target-identifying CRISPR RNA duplex (crRNA and trans-activating crRNA (tracrRNA)) that can be simplified into a single guide RNA (sgRNA). sgRNA sequence can match and target with an 18- to 25-bp DNA sequence, with a required DNA motif termed the protospacer-adjacent motif (PAM) adjacent to the binding site. The most commonly used type of Cas9 is derived from Streptococcus pyogenes, and the PAM sequence is NGG (N represents any nucleotide), while NAG works sporadically with lower efficiency.
In CRISPR-Cas9 system, sgRNA with a general 20 bp custom designed sequence determines target specificity and efficiency. Designing sgRNA is an indispensible part of CRISPR related projects. Of the published tools that enable automated sgRNA design, CRISPR-ERA can provide sgRNA searching approaches for both gene editing and gene regulation applications, and provide additional genome-wide sgRNA library building protocol (Liu et al., 2015). Currently, CRISPR-ERA supports sgRNA design for nine organisms with different kinds of manipulations. It provides a user-friendly webserver to enable sgRNA searching in preassembled databases. The preassembled genome-wide sgRNA databases are built by seeking all targetable sites with patterns of N20NGG. To evaluate the efficiency and specificity of each sgRNA, CRISPR-ERA utilizes criteria summarized from published data, and then computes an efficacy score (E-score) and a specificity score (S-score). Criteria will have a slight change within different kinds of manipulation and organisms.
Equipment
Personal computer for CRISPR-ERA website searching
High performance computing cluster for building genome-wide sgRNA library. Taken genome version hg19 as an example, the minimum storage space is 500 G
Software
CRISPR-ERA (http://crispr-era.stanford.edu/)
USCS genome browser (Kent et al., 2002; http://genome.ucsc.edu/)
Bowtie2 (Langmead et al., 2012; http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
NCBI (https://www.ncbi.nlm.nih.gov/)
Perl scripts (Programming language, https://www.perl.org/)
Shell scripts (Programming language, Command Line Interface shell, https://www.linux.org/)
Procedure
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Category
Molecular Biology > DNA > DNA cloning
Molecular Biology > DNA > Mutagenesis
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2,523 | https://bio-protocol.org/exchange/protocoldetail?id=2523&type=0 | # Bio-Protocol Content
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FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction
Nadezhda S. Sabeva
MB Maria Bykhovskaia
Published: Vol 7, Iss 17, Sep 5, 2017
DOI: 10.21769/BioProtoc.2523 Views: 7993
Edited by: Pengpeng Li
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the Drosophila neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are loaded with the FM1-43 dye, pre-fixed, treated and illuminated to photoconvert the dye, and then processed for conventional electron microscopy. This procedure enables clear identification of stained synaptic vesicles at electron micrographs.
Keywords: Electron microscopy Photoconversion FM1-43 Drosophila Synaptic vesicle Recycling pool Neuromuscular junction
Background
Neuronal transmitters are released via the fusion of synaptic vesicles with the neuronal plasma membrane. Vesicles can fuse spontaneously or in response to an action potential. Subsequently, vesicles become retrieved via endocytosis and recycled. Molecular mechanisms of synaptic vesicle recycling were investigated extensively with the tools of molecular biology, electrophysiology and microscopy (Slepnev and De Camilli, 2000; Sudhof, 2004; Rizzoli and Betz, 2005; Kavalali, 2006). Loading the endocytic marker FM1-43 coupled with the dye photoconversion followed by electron microscopy analysis is a powerful technique that allows the investigation and measurement of the recycling vesicle pools (Harata et al., 2001; Schikorski and Stevens, 2001; Rizzoli and Betz, 2004). Drosophila neuromuscular junction (NMJ) is an advantageous preparation with clearly defined synaptic boutons, which enables rapid generation of lines with mutated synaptic proteins and rigorous evaluation of vesicle recycling pools (Akbergenova and Bykhovskaia, 2009; Denker et al., 2009). A fundamental question in the field of synaptic transmission is whether the evoked and spontaneous transmission utilizes the same recycling pool. To address this question, the recycling pool utilized in the absence of stimulation needs to be measured. This is a challenging problem due to low rates of spontaneous release and recycling. We have developed a protocol for FM1-43 loading followed by the dye photoconversion and EM analysis, which enables rigorous evaluation of recycling pools utilized during spontaneous and evoked transmission at the Drosophila NMJ (Sabeva et al., 2017).
Materials and Reagents
Sample preparation
Gloves
Long sleeve lab coat
Sample bottle with snap cap, size 4 ml (Electron Microscopy Sciences, catalog number: 64250 )
Falcon 35 mm culture plates (Corning, NY)
Drosophila melanogaster fly stocks: Canton S (Bloomington Drosophila Stock Center, catalog ID: FBst1000081) and cpxSH1 (cpx-/-) (Huntwork and Littleton, 2007)
Ca2+-free HL3 saline containing 75 μM advasep-7 (Biotium, catalog number: 70029 ) (see Note 1)
FM1-43 (Thermo Fisher Scientific, InvitrogenTM, catalog number: T35356 ), 10 µM
100 mM NH4Cl (Sigma-Aldrich, catalog number: A9434 ) in HEPES
1.5 mg/ml DAB (Agilent Technologies, DAKO, OEM) in HEPES
Note: The compound is available liquid or tablets in kits.
1% osmium tetroxide (OsO4) prepared in 90 mM cacodylate buffer using the 4% OsO4 stock solution (Electron Microscopy Sciences, catalog number: 19150 )
Ascending acetone series 50, 70, and 90% prepared from 100%
2% uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 ) prepared in water
Acetone, 100% (Sigma-Aldrich, catalog number: 270725 ) dehydrated with molecular sieve dehydrate Fluka (Fluka Analysis, Sigma-Aldrich, catalog number: 270725 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911 )
Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6014 )
Trehalose (Sigma-Aldrich, catalog number: T9531 )
Sucrose (Sigma-Aldrich, catalog number: S1888 )
HEPES-Na salt (Sigma-Aldrich, catalog number: H7006 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16220 )
Sodium cacodylate buffer (Electron Microscopy Sciences, catalog number: 11653 )
HL3 Drosophila saline solution (see Recipes)
Pre-fixative solution (see Recipes and Note 2)
HEPES-buffered saline (see Recipes)
Fixative solution (see Recipes and Note 2)
Sample embedding
ACLAR® 33C Embedding film (Electron Microscopy Sciences, catalog number: 50425-10 )
Tri-Corn beakers: plastic, disposable (Electron Microscopy Sciences, catalog number: 60972 )
Flat Bottom Embedding Capsules (Electron Microscopy Sciences, catalog number: 70021 )
Epon (Embed-812) (Electron Microscopy Sciences, catalog number: 14900 )
Nadic Methyl Anhydride (NMA) (Electron Microscopy Sciences, catalog number: 19000 )
Dodecenyl Succinic Anhydride Specially Distilled (DDSA) (Electron Microscopy Sciences, catalog number: 13710 )
2,4,6-Tri(dimethylaminomethyl) phenol (DMP-30) (Electron Microscopy Sciences, catalog number: 13600 )
ERL-4221 (Electron Microscopy Sciences, catalog number: 15004 )
D.E.R. 736 Epoxy Resin (Used to simplify infiltration in combination with Embed 812) (Electron Microscopy Sciences, catalog number: 13000 )
Nonenyl Succinic Anhydride Modified (NSA) (Electron Microscopy Sciences, catalog number: 19050 )
Dimethylaminoethanol (DMAE) (Electron Microscopy Sciences, catalog number: 13300 )
Embedding Mix A (see Recipes)
Embedding Mix B (see Recipes)
Equipment
Sample preparation
Sylgard 184 (World Precision Instruments, Sarasota, FL)
Fine tweezers (World Precision Instruments, models: #2 and #5 )
Fine scissors (World Precision Instruments, model: 501233 )
Insect Minutien Pins (0.1 mm) (Fine Science Tools, catalog number: 26002-10 )
Dissecting stereoscopic zoom microscope (Nikon Stereozoom Microscope, Nikon Corporation)
A.M.P.I. Master 8 Stimulator (A.M.P.I., model: Master-8 )
Suction electrode filled with HL3
Epifluorescence compound microscope with a filter cube with a long-pass emission filter customized for FM1-43 dye
60x water-immersion objective (ZEISS, Thornwood, NY)
Mercury lamp
Biowave (Ted Pella, Redding, CA)
480 ± 10 bandpass excision filter
PELCO® R1 Single Speed Rotator in the 35° positions (Ted Pella, model: PELCO® R1 )
Fume hood
Sample embedding
Oven Binder (Tuttlingen, Germany)
Ultrathin Sections preparation and Image Capture
Ultramicrotome for ultrathin sectioning (Leica, model: Leica EM UC6 )
Formvar/carbon-coated 2 x 1 mm slot copper grids (Electron Microscopy Sciences, catalog number: FCF-2010-Cu )
Ultra Diatome diamond sectioning knife, wet (Electron Microscopy Sciences, catalog number: 27-US )
UltraTrim Dry Room Temperature Diatome knife, 4.0 mm (Electron Microscopy Sciences, catalog number: UTT-40-R )
Eyelash with handle (Ted Pella, catalog number: 119 )
Double edge stainless steel razor blades (Electron Microscopy Sciences, Hatfield, PA)
Forceps for grids (Electron Microscopy Sciences, Hatfield, PA)
JEOL 100 CX Electron Microscope equipped with Hamamatsu digital camera and AMT software
Software
ImageJ (National Institutes of Health) for image analysis
Adobe Photoshop (Adobe Systems) software for image analysis
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Sabeva, N. and Bykhovskaia, M. (2017). FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction. Bio-protocol 7(17): e2523. DOI: 10.21769/BioProtoc.2523.
Sabeva, N., Cho, W. R., Vasin, A., Gonzalez, A., Littleton, T. J. and Bykhovskaia, Maria. (2017). Complexin mutants reveal partial segregation between recycling pathways that drive evoked and spontaneous neurotransmission. J Neurosci 37: 383-396.
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Category
Neuroscience > Development > Neuron
Cell Biology > Cell imaging > Electron microscopy
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2,524 | https://bio-protocol.org/exchange/protocoldetail?id=2524&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma
Heather A. McCauley
Géraldine Guasch
Published: Vol 7, Iss 17, Sep 5, 2017
DOI: 10.21769/BioProtoc.2524 Views: 8661
Edited by: Nicoletta Cordani
Reviewed by: Shweta Garg
Original Research Article:
The authors used this protocol in 13-Mar 2017
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The authors used this protocol in:
13-Mar 2017
Abstract
Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further functionally profile the cancer stem cells (RNA sequencing and epigenetic analysis), grow them in culture or use them directly in transplantation assays.
Keywords: Cancer stem cells Flow cytometry Cell isolation Squamous cell carcinoma
Background
Tumor recurrence and metastasis is the leading cause of most deaths related to cancer. Malignant tumors may be initiated and maintained by a stem cell population (Nassar and Blanpain, 2016; Bonnet and Dick, 1997), and these cells represent an important therapeutic target to prevent relapse (Baumann et al., 2008). Studies suggest that squamous cell carcinomas are maintained by a subpopulation of tumor cancer stem cells that are resistant to therapy and can initiate tumor recurrence by undergoing self-renewal and differentiation, like normal stem cells, giving rise to proliferating progenitor cells that differentiate and form the bulk of the tumor (Locke et al., 2005; Prince et al., 2007; Malanchi et al., 2008; de Sousa e Melo et al., 2017). In this scenario, tumor cell fate and behavior are determined by the specific combination of changes in genes or their expression that have occurred during tumor development (Wang, 2010). Alternatively, progenitor cells can also acquire mutations that give them the potential to self-renew or can acquire some plasticity that give them cancer stem cell properties (Shimokawa et al., 2017). Regardless of the origin of cancer stem cells, efficient techniques to isolate these cells while maintaining their viability is essential. We have extensively characterized cancer stem cells from anorectal transition zone squamous cell carcinoma which arise spontaneously in the absence of epithelial TGFβ signaling (Keratin14Cre; Tgfbr2flox/flox mice) (Guasch et al., 2007; McCauley and Guasch, 2013; McCauley et al., 2017 ). In this protocol, we describe a method to isolate cancer stem cells from these anorectal transition zone squamous cell carcinomas.
Materials and Reagents
Tissue culture dishes, 100 x 20 mm (Corning, Falcon®, catalog number: 353003 ) and 60 x 15 mm (Corning, Falcon®, catalog number: 353002 )
Sterile disposable scalpel, #21 (Sklar)
50 ml conical tubes (BD, Falcon)
Pipettes (25 ml, 10 ml and 5 ml serological and 1,000 µl, 200 µl, 20 µl and 2 µl pipette tips)
Sterile nylon cell strainers, 70 μm (Fisher Scientific, catalog number: 22-363-548 ) and 40 µm (Fisher Scientific, catalog number: 22-363-547 )
5 ml polystyrene round-bottom tubes with cell strainer caps, 12 x 75 mm style (Corning, Falcon®, catalog number: 352235 )
Sterile screw cap tubes and caps with ‘O’ rings, 1.5 ml (Corning, Axygen®, catalog number: SCT-150-C-S )
Nalgene sterile disposable vacuum filters (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167-0045 )
Healthy and Tumor-bearing mice
For healthy control mice, we used any mouse in the colony that did not express a transgene
We used Keratin14Cre; Tgfbr2flox/flox; R26R-eYFPSTOP-flox-STOP mice, which spontaneously develop anorectal squamous cell carcinoma (Guasch et al., 2007; McCauley and Guasch, 2013; McCauley et al., 2017) in the development of this protocol. All three alleles are available from Jackson Labs (THE JACKSON LABORATORY, catalog number: 018964 , 012603 , and 006148 , respectively)
1x Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14170112 )
Deoxyribonuclease (DNAse) I, from bovine pancreas, 10 mg/ml (Sigma-Aldrich, catalog number: D4263-5VL )
1x phosphate-buffered saline, sterile (1x PBS) (made in-house)
Trypsin-EDTA, 0.25% (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 ) pre-warmed to 37 °C
7-AAD, 0.05 mg/ml (BD, BD Biosciences, catalog number: 559925 )
Antibodies
PE-Cy7 rat-anti-mouse CD11b, clone M1/70, 1/200 dilution (BD, BD Biosciences, catalog number: 561098 , RRID:AB_ 2033994)
PE-Cy7 rat-anti-mouse CD31, clone 390, 1/100 dilution (BD, BD Biosciences, catalog number: 561410 , RRID:AB_10612003)
PE-Cy7 rat-anti-mouse CD45, clone 30-F11, 1/200 dilution (Thermo Fisher Scientific, eBioscienceTM, catalog number: 25-0451-82 , RRID:AB_469625)
PE rat-anti-human CD49f, 1/50 dilution (BD, BD Biosciences, catalog number: 555736 , RRID AB_396079)
Pacific Blue anti-mouse/rat CD29, clone HMb1-1, 1/100 dilution (BioLegend, catalog number: 102224 , RRID:AB_2128079)
Biotin anti-mouse CD34, clone RAM34, 1/50 dilution (Thermo Fisher Scientific, eBioscienceTM, catalog number: 13-0341-85 , RRID:AB_ 466426)
APC Streptavidin, 1/200 dilution (BD, BD Bioscience, catalog number: 554067 , RRID:AB_10050396)
β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
QIAGEN RNeasy Micro kit (QIAGEN, catalog number: 74004 )
Matrigel Matrix Basement Membrane, 5 ml vial (Corning, catalog number: 356234 )
Sterile distilled, deionized water
Collagenase (Sigma-Aldrich, catalog number: C2674-1G )
20% collagenase from Clostridium histolyticum (see Recipes)
Chelated fetal bovine serum (FBS) (see Recipes) (Nowak and Fuchs, 2009)
Chelex, 100 resin, sodium, 200-400 dry mesh, 75-150 µm wet bead (Bio-Rad Laboratories, catalog number: 1422842 )
Epithelial cell culture medium (see Recipes) (Nowak and Fuchs, 2009)
Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient medium (3:1 Mix) without Calcium (Gibco Invitrogen, special order custom powdered media, catalog number: 90-5010EA)
Ham’s F-12 nutrient mixture (Thermo Fisher Scientific, GibcoTM, catalog number: 11765054 )
Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21051024 )
Hydrocortisone (EMD Millipore, catalog number: 3867 )
Cholera toxin (MP Biomedicals, catalog number: 02150005 )
Insulin (Sigma-Aldrich, catalog number: I6634 )
Transferrin (Roche Diagnostics, catalog number: 10652202001 )
3,3’,5-Triiodo-L-thyronine (T3) (Sigma-Aldrich, catalog number: T2877 )
HCl and NaOH for pH adjustment and preparing dilutions of media additives
Staining buffer (2% FBS) (see Recipes)
Equipment
Rocking platform (VWR)
Precision gravity convection incubator set to 37 °C and 5% CO2 (GCA corporation)
Pipet aid (Drummond) and micro-pipettors (Rainin)
Refrigerated centrifuge (Eppendorf, model: 5810 R )
Laminar flow hood (Labgard)
Autoclave (Steris)
4 L beaker (Pyrex)
6 L Erlenmeyer flask (Pyrex)
Glass screw-top bottles, autoclaved (Pyrex)
Graduated cylinders (Nalgene)
Stir plate and stir bars (Fisher Scientific)
pH meter (Fisher Scientific)
Compressed CO2 source (Praxair)
Sterile scissors, Iris Ribbon, 4” (Sklar)
Sterile dissecting forceps, Half-curved 1 mm tip, 4” (George Tiemann & Co)
FACS Aria II, 4 lasers 405/488/561/633 nm (Becton Dickinson) with 130 μm nozzle (BD, model: FACSAriaTM II )
Procedure
Dissociation of squamous cell carcinoma into single-cells
Sacrifice mice according to standard protocol. We use CO2 inhalation followed by cervical dislocation to ensure death has occurred.
Our tumor-bearing mice contain the eYFP reporter allele (Figure 1A). Because of this, we also sacrifice a healthy, wild-type mouse which does not contain a fluorescent reporter and dissociate keratinocytes from the wild-type anorectal transition zone to obtain cells for unstained and single-stained controls. Dissociate cells from the anorectal transition zone of this healthy, wild-type mouse in parallel to dissociation of tumor cells from the tumor-bearing mouse.
Carefully dissect the tumor, ensuring that it is separated from skin, fat, muscle and other contaminating tissue (Figure 1A). Remove any necrotic tissue.
Place the tumor, approximately 0.5 cm2, in 8 ml sterile 1x HBSS in a 10 cm Petri dish and mince using a scalpel until all pieces are uniform and small (Figure 1B).
Add 100 µl 20% collagenase (see Recipes) to dissociate the minced tumor. Incubate for 45 min while shaking at 37 °C. We place a rocking platform within a dry incubator set at speed 3 rpm.
After 45 min, the tumor mixture should be homogeneous and may appear viscous (Figure 1C). Add 8.3 µl DNase I (10 mg/ml) to the minced tumor and incubate for an additional 10 min while shaking at 37 °C. After DNAse I treatment, the viscosity of the tumor mixture should be greatly reduced (Figure 1D).
Figure 1. Dissociation of squamous cell carcinoma. A-A’. Keratin14Cre; Tgfbr2 flox/flox mice develop squamous cell carcinoma at the transition between the anal canal and the rectum. We have crossed these mice into mice containing the R26R-eYFPSTOP-flox-STOP allele such that the resulting Keratin14-positive cells, including the anorectal squamous cell carcinoma, express YFP. B. After removing the skin, fat and rectum from the tumor, mince the tumor into small pieces and incubate in HBSS containing 20% collagenase for 45 min (steps A4-A5). C. After 45 min of collagenase treatment, the tumor mixture will appear viscous (step A6). D. After 10 minutes of DNAse I treatment, the viscosity of the tumor mixture will be greatly reduced (step A6). Figures 1B-1D is adapted from McCauley and Guasch, 2013.
Using a 25 ml pipette, add 10 ml cold 1x PBS to the minced tumor and pipette up and down vigorously 8 to 10 times to further dissociate the tumor mixture. Add the minced tumor to a 50 ml conical tube on ice.
Wash the plate twice with 10-15 ml cold 1x PBS to obtain the maximum number of cells extracted from the tumor and add to the same 50 ml conical tube for each sample dissected.
Centrifuge for 10 min at 2,000 x g at 4 °C.
Carefully aspirate the supernatant, as the cell pellet may be very loose at this step. Using a 10 ml pipette, resuspend the cell pellet with 20 ml cold 1x PBS and add 200 µl chelexed FBS.
Place a 70 µm filter set atop a new 50 ml conical tube. Pre-wet the filter with 1x PBS and filter the tumor cell suspension.
Place the 70 µm filter in a 60 mm culture dish and add 2 ml of pre-warmed 0.25% trypsin-EDTA to the filter to maximize the number of cells extracted from the minced tumor. Incubate the filter for 10 min at 37 °C. This step is crucial as most of the epithelial CD34+ cells will be obtained at this dissociation step.
After 5 min, block the activity of the trypsin by adding 5 ml epithelial cell culture media (see Recipes) containing serum to the filter. Mix well the cells with a 5 ml pipette to completely dissociate any clumps. Add the filtered trypsinized cells to the filtered cells in the same 50 ml conical tube from step 11. Wash the 60 mm plate twice with another 5 ml media to maximize the number of cells obtained.
Place a 40 µm filter set atop a new 50 ml conical tube and pre-wet with epithelial cell culture media. Using a 5 ml pipette, pass the cells through the 40 µm filter.
Centrifuge for 10 min at 200 x g at 4 °C.
Aspirate the supernatant and resuspend the cell pellet in 1 ml staining buffer (see Recipes) in PBS to create a single-cell suspension.
Separation of cancer stem cells from squamous cell carcinoma
Filter the single-cell suspension through pre-blocked FACS tubes with a cell strainer cap. Pre-wet the cell strainer with PBS before applying the cell suspension to maximize cell number obtained.
Note: To pre-block FACS tubes, we fill each tube that will be used, including those for single-stained and unstained controls, with FBS, allowing the FBS to coat the walls of the tubes. We then decant the FBS and reserve for reuse, and fill each blocked tube with 1x PBS until needed.
Aliquot approximately 100 µl of cell suspension to the appropriate number of pre-blocked FACS tubes for unstained and single-stained controls. For our experiments:
Wild-type anorectal transition zone cells are split into:
Unstained control
7AAD control (reserve until immediately before sort)
PE-Cy7 control
PE control
Pacific Blue control
APC control
YFP+ tumor cells are split into:
YFP control (100 µl)
The remainder of the cell suspension (approximately 1 ml) contains all colors and is the sample which will be sorted
Add the appropriate antibody to each tube.
Incubate the cells on ice and in the dark for 30 min. Tap the tubes every 5-10 min to disturb the cells from settling at the bottom of the tube.
Fill each tube with 2% FBS to wash.
Centrifuge for 5 min at 200 x g.
Dump the supernatant into a waste container. Approximately 200 µl of buffer, containing the cell pellet, should remain at the bottom of the tube.
We found that using the biotin-CD34 with the SAV-APC optimized the signal for the rare CD34+ cells within our tumors. For the APC control tube and the sample to be sorted, incubate with secondary antibody SAV-APC for 20 min on ice, protected from light.
Note: Reserve the remainder of the control tubes on ice and protected from light during this time.
Fill these tubes with 2% FBS to wash.
Centrifuge for 5 min at 200 x g.
Dump the supernatant into a waste container. Approximately 200 µl of buffer, containing the cell pellet, should remain at the bottom of the tube.
Resuspend the cell pellet of the tumor sample with 300-800 µl 2% FBS. The volume should be adjusted to account for the size of the cell pellet to optimize flow rate on the cytometer. Keep in mind that a concentrated sample can easily be diluted.
Bring the 7AAD with the sample to the flow cytometer. We prefer to add the 7AAD to the sample immediately before sorting to minimize cell death due to toxicity. We found 20 µl per million cells to be sufficient to label dead cells.
Record at least 10,000 events from the unstained and single-color controls to enable compensation.
See Figure 2 for an example of our gating strategy to isolate epithelial CD34+ cancer stem cells. The population hierarchy is:
Forward scatter (A) versus side scatter (A).
Side Scatter (H) versus Side Scatter (W) doublet discrimination.
Forward Scatter (H) versus Forward Scatter (W) doublet discrimination.
7AAD negative (live), PE-Cy7 negative (CD11b, CD31, CD45 staining).
YFP positive.
PE positive (a6-integrin), Pacific Blue positive (b1-integrin).
APC negative and positive (CD34).
Figure 2. Gating strategy to isolate epithelial CD34+ cancer stem cells from Tgfbr2-deficient tumors. After dissociation and filtering into a single cell suspension, cells are subjected to fluorescence-activated cell sorting to isolate the rare epithelial CD34+ cancer stem cells. Cells of the appropriate size and granularity are included in P1, and doublets are excluded by the SSC and FSC gates. Live cells are selected by 7AAD exclusion, and CD11b+, CD31+ and CD45+ cells are excluded in the DUMP channel by PE-Cy7 staining. LIVE cells which express YFP under control of the Keratin 14 promoter are then further purified by α6- and β1-integrin expression (PE and Pacific Blue, respectively). Of these YFP+α6+β1+ cells, a pure population of CD34 (APC+) cancer stem cells can then be isolated.
We have successfully isolated live cells for RNA extraction, for returning to culture, and for direct transplantation into recipient mice. Depending on the size of the initial tumor, the efficiency of dissociation, and individual composition of the tumor, we typically obtain between 25,000 and 250,000 live, YFP+, a6-integrin+ b1-integrin+ tumor cells from a tumor approximately 0.5 cm3, and from that tumor population, we typically obtain between 2,000-10,000 CD34+ cancer stem cells.
For RNA extraction, collect 300 to 10,000 cells directly into 350 µl lysis buffer containing 3.5 µl β-mercaptoethanol in screw-top tubes. Vortex the tubes immediately after sorting and store at -80 °C until RNA extraction. We prefer the QIAGEN RNeasy Micro kit and use the buffer RLT from the kit.
For return to culture, collect as many cells as possible in 500 µl epithelial cell culture media in a screw-top tube. Centrifuge tubes at 200 x g for 5 min and very carefully aspirate supernatant. Resuspend in epithelial cell culture media and plate. We found that cells did not survive when plating directly on plastic. We prefer to plate cells on irradiated mouse fibroblasts to improve colony formation and survival. We found that plating less than 1,000 cells did not allow for cell survival.
For direct transplantation, collect as many cells as possible in 500 µl epithelial cell culture media without serum in a screw-top tube. The serum generates bubbles which negatively affect the resuspension in 30% Matrigel. Centrifuge tubes and very carefully aspirate the supernatant. Resuspend in 30% Matrigel, and keep tubes on ice until transplantation (see McCauley and Guasch [2013] for the detailed protocol of the orthotopic transplantation). We have successfully generated tumors by transplanting as few as 100 CD34+ cells. Transplanting larger numbers of cells will accelerate tumor formation.
Data analysis
Because the isolation of rare cells requires inclusion and exclusion of a number of markers, it is imperative to have unstained and single-stained controls for effective compensation of spectral overlap.
The frequency of epithelial CD34+ cells ranged from 7% to 22%. This is due to the heterogeneity of the squamous cell carcinoma from mouse to mouse. It is therefore recommended to perform at least six biological repeats within each experimental group.
Notes
All experiments were approved by the Cincinnati Children’s Hospital Research Foundation Institutional Animal Care and Use Committee (protocol number 1D10087) and in agreement with European and national regulation (protocol number 4572), and carried out using standard procedures.
Trypsinizing the filter is a crucial step that can drastically impact the efficiency of CD34+ cancer stem cells isolated, as the cells may remain in clumps on top of the filter and be unintentionally discarded. Be sure to use pre-warmed 0.25% trypsin-EDTA, to incubate for the full 10:00 at 37 °C, and to vigorously pipet the trypsinized cells to ensure proper dissociation and maximal number of cells obtained.
It is possible to store the tumor samples in media containing serum overnight at 4 °C and perform the cell isolation the next day. Cell mortality will be increased but the overall percentage of cancer stem cells will not be altered.
FBS lots need to be tested for optimal growth of the epithelial cells.
Because epithelial cells are very sensitive to calcium, chelating the FBS is essential.
While it is possible to collect sorted cells using a 100 μm nozzle, we recommend using a 130 μm nozzle to improve the survival of the large epithelial cells post-sort.
Recipes
20% collagenase from Clostridium histolyticum
Prepare a 20% stock by dissolving 1 g of powdered collagenase in 5 ml 1x sterile phosphate-buffered saline
Aliquot 250 μl into Eppendorf tubes and store at -20 °C to avoid repeated freezing and thawing that will decrease enzyme activity
Chelated fetal bovine serum (FBS), prepared as described in (Nowak and Fuchs, 2009)
Day 1
Add 400 g of dry Chelex to a 4 L beaker with a stir bar. Add distilled water to a total volume of 4 L. Cover and stir continuously
Adjust pH to 7.4 using 10 N HCl. Stir for 20 min, readjust pH with 10 N HCl, and repeat as needed until pH remains stable for more than 20 min
Place the beaker at 4 °C overnight to allow the Chelex to form a compact pellet
Day 2
Carefully decant H2O. Add fresh H2O to 4 L
Adjust the pH to 7.4 as in Day 1
Place the beaker at 4 °C for 1 h to allow the Chelex to form a compact pellet
Carefully decant H2O
Slowly add two 500 ml bottles of characterized or defined fetal bovine serum to the Chelex
Stir slowly at 4 °C for 1 h. Try to minimize bubbles
Place the beaker at 4 °C for 1 h to allow the Chelex to form a compact pellet
Decant the serum into a 1 L glass bottle and filter the serum through a Nalgene bottle top filter under sterile conditions
Store unused FBS at 20 °C or use immediately to make E media without calcium
Epithelial cell culture media, prepared as described in (Nowak and Fuchs, 2009)
In a 6 L Erlenmeyer flask, combine six packets of Gibco Invitrogen customized DMEM:F12 (3:1) without calcium with distilled water to reach a final volume of 5.5 L
Add 18.42 g of sodium bicarbonate, 2.85 g of L-glutamine, and 60 ml of 100x penicillin-streptomycin solution
Adjust pH to 7.2 using 10 N HCl and adjust the volume to 6 L with H2O
Apply compressed CO2 to the media for 15 min. The media should reach an amber color
Prepare the following cocktail of media additives:
20 ml of 5 mg/ml insulin, dissolved in 0.1 N HCl
20 ml of 5 mg/ml transferrin, dissolved in sterile PBS
20 ml of 2 x 10-8 M T3, dissolved to 2 x 10-4 in 0.02 N NaOH then further diluted to final concentration in 1x PBS
140 ml 1x PBS
Filter sterilize and store in 37.5 ml aliquots at -20 °C
Add 75 ml of the above cocktail, 750 ml 10-6 M cholera toxin (dissolved in water), and 750 ml 4 mg/ml hydrocortisone to 1 L of chelated FBS
Produce final 15% FBS media in 1 L batches by combining 850 ml of the DMEM:F12 media base from step 2 with 150 ml of the supplemented chelated FBS and sterilize using a Nalgene bottle top filter
Media can be stored in 250 or 500 ml bottles at 20 °C
Staining buffer (2% FBS)
1 ml chelexed FBS
49 ml 1x PBS
Note: Make fresh for each use, and keep on ice.
Acknowledgments
The protocol to dissociate the tumor is based on our original protocol published in Methods Mol Biol (McCauley and Guasch, 2013), with minor modifications. The protocol to make chelexed FBS and E media are abbreviated here, but made exactly as described in (Nowak and Fuchs, 2009). An abbreviated description of the flow cytometry protocol appeared in eLife (McCauley et al., 2017). We would like to acknowledge the assistance of the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center (supported in part by P30 DK07839) and the Flow Cytometry Core at the CRCM, FRANCE. The protocol related to this work was supported by grants from the V Foundation, the Sidney Kimmel Foundation and in part from the Foundation ARC pour la recherche sur le cancer (GG).
References
Baumann, M., Krause, M. and Hill, R. (2008). Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 8(7): 545-554.
Bonnet, D. and Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3(7): 730-737.
de Sousa e Melo, F., Kurtova, A. V., Harnoss, J. M., Kljavin, N., Hoeck, J. D., Hung, J., Anderson, J. E., Storm, E. E., Modrusan, Z., Koeppen, H., Dijkgraaf, G. J., Piskol, R. and de Sauvage, F. J. (2017). A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer. Nature 543(7647): 676-680.
Guasch, G., Schober, M., Pasolli, H. A., Conn, E. B., Polak, L. and Fuchs, E. (2007). Loss of TGFβ signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia. Cancer Cell 12(4): 313-327.
Kreso, A. and Dick, J. E. (2014). Evolution of the cancer stem cell model. Cell Stem Cell 14(3): 275-91.
Locke, M., Heywood, M., Fawell, S. and Mackenzie, I. C. (2005). Retention of intrinsic stem cell hierarchies in carcinoma-derived cell lines. Cancer Res 65(19): 8944-8950.
Malanchi, I., Peinado, H., Kassen, D., Hussenet, T., Metzger, D., Chambon, P., Huber, M., Hohl, D., Cano, A., Birchmeier, W. and Huelsken, J. (2008). Cutaneous cancer stem cell maintenance is dependent on β-catenin signalling. Nature 452(7187): 650-653.
McCauley, H. A., Chevrier, V., Birnbaum, D. and Guasch, G. (2017). De-repression of the RAC activator ELMO1 in cancer stem cells drives progression of TGFβ-deficient squamous cell carcinoma from transition zones. Elife 6.
McCauley, H. A. and Guasch, G. (2013). Serial orthotopic transplantation of epithelial tumors in single-cell suspension. Methods Mol Biol 1035: 231-245.
Nassar, D. and Blanpain, C. (2016). Cancer stem cells: Basic concepts and therapeutic implications. Annu Rev Pathol 11: 47-76.
Nowak, J. A. and Fuchs, E. (2009). Isolation and culture of epithelial stem cells. Methods Mol Biol 482: 215-232.
Prince, M. E., Sivanandan, R., Kaczorowski, A., Wolf, G. T., Kaplan, M. J., Dalerba, P., Weissman, I. L., Clarke, M. F. and Ailles, L. E. (2007). Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 104(3): 973-978.
Shimokawa, M., Ohta, Y., Nishikori, S., Matano, M., Takano, A., Fujii, M., Date, S., Sugimoto, S., Kanai, T. and Sato, T. (2017). Visualization and targeting of LGR5+ human colon cancer stem cells. Nature 545(7653): 187-192.
Wang, J. C. (2010). Good cells gone bad: the cellular origins of cancer. Trends Mol Med 16(3): 145-151.
Copyright: McCauley and Guasch. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
McCauley, H. A. and Guasch, G. (2017). Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma. Bio-protocol 7(17): e2524. DOI: 10.21769/BioProtoc.2524.
McCauley, H. A., Chevrier, V., Birnbaum, D. and Guasch, G. (2017). De-repression of the RAC activator ELMO1 in cancer stem cells drives progression of TGFβ-deficient squamous cell carcinoma from transition zones. Elife 6.
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Cancer Biology > Cancer stem cell > Tumor formation
Cancer Biology > Cancer stem cell > Cell biology assays
Cell Biology > Cell isolation and culture > Cell isolation
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2,525 | https://bio-protocol.org/exchange/protocoldetail?id=2525&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation
Steven Boeynaems
MD Mathias De Decker
PT Peter Tompa
LB Ludo Van Den Bosch
Published: Vol 7, Iss 17, Sep 5, 2017
DOI: 10.21769/BioProtoc.2525 Views: 12883
Edited by: Nicoletta Cordani
Reviewed by: Weiyan JiaMirko Messa
Original Research Article:
The authors used this protocol in Mar 2017
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Original research article
The authors used this protocol in:
Mar 2017
Abstract
Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems et al., 2017).
Keywords: LLPS Amyotrophic lateral sclerosis Peptides Arginine Phase separation Mass spec C9orf72 Stress granule
Background
Arginine-rich disordered domains are often found in RNA binding proteins, including the ones associated with neurodegenerative diseases (e.g., FUS, FMRP, hnRNPA1) (Varadi et al., 2015; Boeynaems et al., 2017). Also toxic arginine-rich repeat peptides (i.e., polyGR and polyPR) are produced in amyotrophic lateral sclerosis patients carrying the C9orf72 repeat expansion (Kwon et al., 2014; Mizielinska et al., 2014; Varadi et al., 2015; Boeynaems et al., 2016). While phase separation of uncharged low complexity domains had been studied before (Kato et al., 2012; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Patel et al., 2015), we developed this protocol to test whether arginine-rich domains could also contribute to phase separation. A schematic representation of the protocol can be seen in Figure 1.
Figure 1. Workflow of protein droplet formation and analysis. A diffuse arginine-rich peptide solution can be induced to undergo liquid-liquid phase separation by addition of RNA or molecular crowder (PEG). Resulting droplets can be further analyzed by fluorescence microscopy and FRAP.
Materials and Reagents
Pipette tips
Amicon® Ultra–0.5 ml centrifugal filters 3K (Merck, catalog number: UFC500396 )
Cell counting slides (Bio-Rad Laboratories, catalog number: 1450015 )
Clear adhesive tape
Custom 20 to 60 amino acid peptides with free carboxy and amino-termini (Pepscan)
Custom Alexa labeled RNA 30 ribonucleotide oligomers (IDT)
MilliQ water
Alexa Fluor® Protein Labeling Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: A10235 )
PEG300 (Sigma-Aldrich, catalog number: 81160 )
Note: This product has been discontinued.
Potassium phosphate monobasic (KH2PO4)
Potassium phosphate dibasic (K2HPO4)
Polyuridylic acid potassium salt (Sigma-Aldrich, catalog number: P9528 )
Clear nail varnish
10x potassium buffer, pH 7 (see Recipes)
Equipment
Pipette
Bench-top centrifuge (Eppendorf, models: 5430 and 5810 R )
trUView cuvettes (Bio-Rad Laboratories, catalog number: 1702510 )
Note: This product has been discontinued.
SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, model: SmartSpec Plus )
LSM 780 Meta NLO confocal microscope (ZEISS, model: LSM 780 NLO ) with 20x long range objective (ZEISS, model: LD Plan-Neofluor 20x/0.4 Corr Ph2 M27, catalog number: 421351-9970-000 )
Software
Zen software (ZEISS)
GraphPad Prism (GraphPad)
Excel (Microsoft)
Procedure
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How to cite：Boeynaems, S., De Decker, M., Tompa, P. and Van Den Bosch, L. (2017). Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation. Bio-protocol 7(17): e2525. DOI: 10.21769/BioProtoc.2525.
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Category
Neuroscience > Nervous system disorders > Blood brain barrier
Biochemistry > Protein > Isolation and purification
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2,526 | https://bio-protocol.org/exchange/protocoldetail?id=2526&type=0 | # Bio-Protocol Content
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Peer-reviewed
Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells
HD Huy Nguyen Duc
Xiaojun Ren
Published: Vol 7, Iss 17, Sep 5, 2017
DOI: 10.21769/BioProtoc.2526 Views: 10953
Edited by: Arsalan Daudi
Original Research Article:
The authors used this protocol in 18-Oct 2016
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18-Oct 2016
Abstract
HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).
Keywords: HaloTag Live-cell single-molecule imaging Polycomb Cbx Epigenetics Janelia FluorTM dye
Background
Molecular processes of living organisms are intrinsically dynamics. Direct observation of the molecular processes in living cells is critical for quantitatively understanding of how biological systems function. Recent advances in fluorescence microscopy and fluorescent labelling enable to visualize trajectories of individually single molecules in living cells, providing insights about dynamic interactions and assemblies of biological molecules (Kusumi et al., 2014; Liu et al., 2015; Tatavosian et al., 2015; Cuvier and Fierz, 2017). Specific labelling of biomolecules with fluorophores is the key for fluorescence single-molecule imaging. HaloTag is self-labeling tag proteins that can be coupled to synthetic dyes in living cells (Los et al., 2008). The reaction occurs rapidly in living cells and the formed covalent bond is specific and irreversible. This technique has been utilized to study the genetic information flow in vivo, and to measure the kinetic of gene regulation in living mammalian cells (Liu et al., 2015; Zheng and Lavis, 2017). Janelia FluorTM dyes, such as Janelia FluorTM 549 (JF549), are bright and photostable fluorescent HaloTag ligands (Grimm et al., 2015). This protocol describes how to label HaloTag-Cbx proteins with JF549 for live-cell single-molecule imaging, which was developed in the recent publication (Zhen et al., 2016).
Materials and Reagents
Pipette tips (BioExpress, catalog number: P-1236-200)
Manufacturer: Biotix, catalog number: P-1236-200CS .
35 mm glass bottom dish made in the laboratory (see Video 1 for making glass-bottom dishes)
Video 1. Making glass-bottom dishes. The video elaborates how to make glass-bottom dishes for live-cell single-molecule imaging.
Cell lines used in protocol: mouse embryonic stem cells and HEK293T cells
Janelia Fluor 549 dye (JF549) provided by Dr. Luke D. Lavis (Janelia Research Campus, Howard Hughes Medical Institute)
Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537-500ML )
Gelatin (Sigma-Aldrich, catalog number: G1890-100G )
DMEM (Sigma-Aldrich, catalog number: D5796 )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0926 )
Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
β-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985023 )
Non-essential amino acids (Thermo Fisher Scientific, catalog number: 1114050 )
Leukemia inhibitor factor (LIF, made in the laboratory)
FluoroBrite DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: A1896701 )
ES cell medium (see Recipes)
Live-cell imaging medium (see Recipes)
Equipment
Single channel pipette (BioExpress, Kaysville, USA)
Heater controller (Warner Instruments, catalog number: TC-324 )
Microscope (Manual Microscopy) (ZEISS, model: Axio Observer D1 )
Alpha Plan-Apochromatic 100/1.46 NA Oil-immersion Objective (ZEISS, Germany)
Evolve 512 x 512 EMCCD camera (Photometrics, Tucson, USA)
Solid state laser (Intelligent Imaging Innovations, 3i LaserStack with Fiber)
Software
Slidebook 6.0 software (Intelligent Imaging Innovations, Denver, Colorado)
MATLAB R2015a (8.5.0.197613) (MathWorks, Natick, USA)
U-track 2.0 (Danusar Lab, UT South Western Medical Center, Dallas, USA)
Procedure
Trypsinize 70-90% confluent cells (mouse embryonic stem cells or HEK293 cells) of 100 mm plate stably expressing HaloTag-Cbx proteins (we recommend 0.6 ml of 0.05% trypsin-EDTA [1x] for 60 mm plate and 1.5 ml for 100 mm plate).
Seed 20% of cells to 35 mm glass-bottom dish coated by Gelatin overnight (Note 1) (see Video 2 for gelatinization).
Video 2. Gelatinization of glass-bottom dishes. The video describes how to gelatinize glass-bottom dishes before seeding cells.
Following by overnight culture, the final confluency of the cells before was between 80-90%. Several concentrations (5 nM, 15 nM, and 30 nM) of JF549 are used to incubate with cells for 15 min at 37 °C in 5% CO2 (Notes 2 and 3) (see Video 3 for adding the JF549 dye).
Video 3. Adding JF549 dyes to cells
Gently wash cells with ES medium (see Recipes) once and incubate in ES medium for 30 min at 37 °C and 5% CO2 (see Video 4 for washing cells).
Video 4. Washing cells with ES cell medium
Replace ES medium with live-cell imaging medium (see Recipes) (see Video 5 for adding live-cell imaging medium).
Video 5. Replacing ES cell medium with live-cell imaging medium
Maintain 37 °C conditions during imaging by using a heater controller. Each plate should be imaged for the maximum of 1.5 h after placing on the microscope (see Video 6 for placing dishes on objective).
Video 6. Placing dishes on objective
The number of individual fluorescent spot per nucleus should be between 10-50 spots, controlled by adjusting the JF549 dye concentrations (Notes 4 and 5) (see Video 7 for single-molecule imaging).
Video 7. Single-molecule imaging. The video describes how to image individual HaloTag-Cbx proteins within living cells.
Movies are then uploaded to u-track 2.0. Each cell is cropped from the larger movie. Cropped movies are processed (Note 6).
Data analysis
Our data were analyzed using MATLAB with u-track 2.0 plug-in, detail guide can be found at http://www.utsouthwestern.edu/labs/danuser/software/ (the software and pdf file guide are included in the download).
Representative images and movies can be found in Zhen et al., 2016.
Notes
Imaging dishes should be gelatinized overnight.
We recommend using pipettes, not suction, when removing medium from imaging dishes.
When add medium to the cells in imaging dishes, we recommend tilting plate at an angle, slowly add medium to the lower edge and let the medium reach the cells slowly.
The TIRF angle used is unique for individual cells and should be adjusted to achieve the best possible movies.
To obtain representative movies, the focus of the movie should be at the middle layer of the nucleus. This can be recognized by the deeper black area (this can be achieved by adjusting the angle). Bright field can be used to check the nucleus area.
In u-track 2.0, multiple movies can be loaded and processed. After being processed, each movie should be checked to ensure that cells should be no drift and rotation.
Recipes
ES cell medium
DMEM
15% FBS
2 mM glutamine
100 U/ml penicillin-streptomycin
0.1 mM β-mercaptoethanol
1,000 U/ml LIF
0.1 mM non-essential amino acids
Live-cell imaging medium
FluoroBrite DMEM supplemented with 15% FBS
2 mM glutamine
100 U/ml penicillin-streptomycin
0.1 mM β-mercaptoethanol
1,000 U/ml LIF
0.1 mM non-essential amino acids
Acknowledgments
This work was supported, in whole or in part, by the National Cancer Institute of the National Institutes of Health under Award Number R03CA191443 (to XR). This work was also supported by grants from the CU-Denver Office Research Service (to XR) and the American Cancer Society Grant IRG 57-001-53 subaward (to XR). This protocol was originally developed in Zhen et al., 2016.
References
Cuvier, O. and Fierz, B. (2017). Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells. Nat Rev Genet.
Grimm, J. B., English, B. P., Chen, J., Slaughter, J. P., Zhang, Z., Revyakin, A., Patel, R., Macklin, J. J., Normanno, D., Singer, R. H., Lionnet, T. and Lavis, L. D. (2015). A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12(3): 244-250, 243 p following 250.
Kusumi, A., Tsunoyama, T. A., Hirosawa, K. M., Kasai, R. S. and Fujiwara, T. K. (2014). Tracking single molecules at work in living cells. Nat Chem Biol 10(7): 524-532.
Liu, Z., Lavis, L. D. and Betzig, E. (2015). Imaging live-cell dynamics and structure at the single-molecule level. Mol Cell 58(4): 644-659.
Los, G. V., Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana, R. F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D. H., Bulleit, R. F. and Wood, K. V. (2008). HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6): 373-382.
Tatavosian, R., Zhen, C. Y. and Ren, X. J. (2015). Single-molecule fluorescence microscopy methods in chromatin biology. Acs Sym Ser 1215:129-136.
Zhen, C. Y., Tatavosian, R., Huynh, T. N., Duc, H. N., Das, R., Kokotovic, M., Grimm, J. B., Lavis, L. D., Lee, J., Mejia, F. J., Li, Y., Yao, T. and Ren, X. (2016). Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5.
Zheng, Q. and Lavis, L. D. (2017). Development of photostable fluorophores for molecular imaging. Curr Opin Chem Biol 39: 32-38.
Copyright: Duc and Ren . This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Duc, H. N. and Ren, X. (2017). Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells. Bio-protocol 7(17): e2526. DOI: 10.21769/BioProtoc.2526.
Zhen, C. Y., Tatavosian, R., Huynh, T. N., Duc, H. N., Das, R., Kokotovic, M., Grimm, J. B., Lavis, L. D., Lee, J., Mejia, F. J., Li, Y., Yao, T. and Ren, X. (2016). Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5.
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Category
Developmental Biology > Cell signaling
Biochemistry > Protein > Labeling
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2,527 | https://bio-protocol.org/exchange/protocoldetail?id=2527&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Sterol Analysis in Kluyveromyces lactis
YG Yvetta Gbelska
NH Nora Toth Hervay
MM Marcela Morvova
AK Alexandra Konecna
Published: Vol 7, Iss 17, Sep 5, 2017
DOI: 10.21769/BioProtoc.2527 Views: 6576
Edited by: Yanjie Li
Reviewed by: Pierre-Damien Denechaud
Original Research Article:
The authors used this protocol in Dec 2016
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Dec 2016
Abstract
Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have been also employed to identify essential steps in the pathway against which antifungals might be developed. We present here a protocol for the isolation of non-saponifiable lipids (sterols) from Kluyveromyces lactis yeast cells and a chromatographic method for quantitative analysis of sterols in lipid extracts (HPLC) that can be performed in laboratories with standard equipment.
Keywords: Ergosterol biosynthesis Kluyveromyces lactis Lipid extraction High-performance liquid chromatography
Background
Ergosterol, the primary membrane sterol found in yeast cells, serves a structural role in cellular membranes similar to that of cholesterol in mammalian systems. Sterols have been shown to be responsible for a number of important physical characteristics of membranes by affecting rigidity, fluidity and permeability of membranes. Through their interactions with phospholipids and sphingolipids, sterols are proposed to maintain the lateral heterogeneity of the protein and lipid distribution in the plasma membrane because of their putative role in inducing microdomains called lipid rafts (Dupont et al., 2011; Souza et al., 2011). Sterol biosynthesis in yeast is an energy-expensive, multistep aerobic process, requiring heme and molecular oxygen. Ergosterol and its biosynthetic steps are the major targets for antifungal compounds which have minor effects on cholesterol synthesis of the host organism (Daum et al., 1998). Both ergosterol and some of its biosynthetic intermediates (squalene, 7-dehydrocholesterol) belong to chemicals with a direct positive appeal to people. Therefore a simple and reliable method for sterol isolation is highly rewarding (Valachovic and Hapala, 2017). In this protocol, we describe the analytical method for sterol isolation used for determination of sterol profile in yeast cells.
Materials and Reagents
Pipette tips (Eppendorf, GBO)
Inoculation loop
15-ml polypropylene centrifuge tubes (Corning, catalog number: 430791 )
Acid-washed glass beads, diameter 0.45 mm (Sigma-Aldrich, catalog number: G8772 ) (see Note 1)
Glass Pasteur pipette (Sigma-Aldrich, catalog number: Z628018 )
20 ml Pyrex® glass tubes with Teflon cups (Corning) (Sigma-Aldrich, catalog number: Z653527) (see Note 1)
Manufacturer: Pyrex, catalog number: 1622/10M .
Kluyveromyces lactis yeast strain to be analysed
n-Hexane (anhydrous 95%) (Sigma-Aldrich, catalog number: 439177 ) (see Note 1)
HPLC standard ergosterol (purity ≥ 95%) (Sigma-Aldrich, catalog number: 45480 ); 0.3 mg/ml stock solution in methanol
Yeast extract (Biolife Italiana, catalog number: 4122202 )
Bacto peptone (Biolife Italiana, catalog number: 4122592 )
D-Glucose (Biolife Italiana, catalog number: 4125012 )
Potassium hydroxide (KOH) pellets (Merck, catalog number: 1050331000 )
Methanol (CHROMASOLVTM for HPLC, ≥ 99.9%) (Honeywell, catalog number: 24229 )
Water (CHROMASOLV® Plus, for HPLC) (Sigma-Aldrich, catalog number: V270733 )
Note: This product has been discontinued.
YEPD rich growth medium (see Recipes)
Methanolic KOH solution (60% KOH, 50% methanol) (see Note 2 and Recipes)
Equipment
Pipettes (Eppendorf, HTL)
Incubation shaker Multitron Standard (Infors HT, Bottmingen, Switzerland)
Haemocytometer
Centrifuge 5804R (Eppendorf, model: 5804 R )
High speed Vortex-Genie 2 (Scientific Industries, model: Digital Vortex-Genie 2 , catalog number: SI-A246)
Automatic sampler (Shimadzu Scientific Instruments, model: SIL-20AC )
HPLC instrument (Shimadzu Scientific Instruments, model: Prominence 20A ) equipped with reversed phase C18 column (Ascentis® Express C18, particle size 5 μm, column size 3 x 150 mm, Supelco), UV-Vis detector (Shimadzu Scientific Instruments, model: SPD-20A )
FastPrep® cell homogenize (MP Biomedicals, model: FastPrep®-24, catalog number: 116004500 )
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Gbelska, Y., Toth Hervay, N., Morvova, M. and Konecna, A. (2017). Sterol Analysis in Kluyveromyces lactis. Bio-protocol 7(17): e2527. DOI: 10.21769/BioProtoc.2527.
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Category
Microbiology > Microbial metabolism > Lipid
Biochemistry > Lipid > Lipid measurement
Biochemistry > Lipid > Lipid isolation
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2,528 | https://bio-protocol.org/exchange/protocoldetail?id=2528&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling
JP Julia Petschnigg
RK Robin Ketteler
Published: Vol 7, Iss 17, Sep 5, 2017
DOI: 10.21769/BioProtoc.2528 Views: 7511
Edited by: Ralph Bottcher
Reviewed by: ilgen MenderTomas Aparicio
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).
Keywords: Grb2 EGFR High-content imaging cDNA si/shRNA Cancer signaling
Background
Signal transduction by growth factor receptors is essential for cells to maintain proper function and thus requires tight control. Signal transduction by growth factor receptors is initiated by binding of an external ligand (e.g., Epidermal Growth Factor, EGF) to a transmembrane receptor such as the Epidermal Growth Factor Receptor (EGFR) and activation of downstream signaling cascades (Yao et al., 2015). A key regulator of EGFR-signaling is Growth Factor Receptor-bound protein 2 (Grb2), which is composed of an internal SH2 (Src homology 2) domain flanked by two SH3 domains. Grb2 binds to activated growth factor receptors at phosphorylated tyrosine residues through its SH2 domain, thus coupling receptor activation to SOS-Ras-MAPK (Mitogen-activated protein kinase) signaling cascades. The composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along multiple pathways. Mutations in signaling pathways frequently lead to the development of cancer. Hence, in order to better understand how aberrant signaling can lead to disease, it is important to identify novel signaling molecules in growth factor signaling. To accomplish this, we used the previously established microscopy-based GFP-Grb2 translocation assay that monitors the translocation of cytosolic GFP-tagged Grb2 to subcellular compartments upon expression of a cDNA library (Figure 1). We used this technique to identify novel proteins that can lead to translocation of GFP-Grb2 when overexpressed and in a second stage tested whether these proteins play a role in EGFR-signaling (Petschnigg et al., 2017). Examples that lead to punctate structures include TACC3, a novel EGFR-interactor, and AMPH (Figure 3). TACC3 led to induction of large GFP-Grb2 puncta, whereas AMPH results in formation of multiple small spots (Figure 3), pointing at potentially different mechanisms of those proteins in EGFR-signaling. In our recent study, we further characterized TACC3 and showed that TACC3 specifically binds to oncogenic EGFR variants and showed that TACC3 enhances EGFR-stability at the cell surface and increases EGFR-mediated signaling. The GFP-Grb2 assay can be expanded to multiple more applications. A siRNA/shRNA or CRISPR library could be co-expressed with GFP-Grb2 and translocation subsequently observed following EGF stimulation. As EGF stimulation would sequester GFP-Grb2 to endosomal structures and the plasma membrane, translocation from there upon siRNA/shRNA knockdown or CRISPR knockout could point at possible factors that ablate EGFR-Grb2 interactions and signaling. In a similar way, small molecule compound screens could be done to test for drugs that can specifically disrupt EGFR-Grb2 interactions upon EGF-stimulation (Figure 1). Other options could be to use mutated Grb2-variants that fail to bind to EGFR or other binding partners, thus the assay can give insight into which gene (when overexpressed or knock-downed) or which drug has an influence on specific binding domains of Grb2.
Figure 1. Schematic overview of the Grb2 translocation assay. Under non-stimulated or basal conditions, GFP-tagged Grb2 is mostly found in the cytosol (A), but can be recruited to localized interaction partners such as activated or endocytosed Epidermal Growth Factor Receptor, EGFR. Expression of a cDNA expression plasmid can lead to relocalization of GFP-Grb2 to the plasma membrane, endosomal structures or other sub-cellular locations by binding to Grb2 interaction partners or activation of Grb2-dependent cellular pathways (B). Stimulation with EGF recruits Grb2 to phosphotylated EGFR (C). Small molecule compounds or si/shRNA libraries can help identify genes or drugs that can disrupt Grb2 binding to the EGFR, impairing the recruitment to the receptor and thus predominant cytosolic localization of GFP-Grb2 (D). Since Grb2 can interact with other growth factor receptors as well, the assay can be adapted to monitor interaction/recruitment to other receptors as well.
Materials and Reagents
Pipette tips (any standard sterile tips can be used, either non-filtered or filtered)
ViewPlate-96 Black, Optically Clear Bottom (PerkinElmer, catalog number: 6005225 )
Tissue culture plates: Nunc cell culture Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 172931 )
0.22 µm filter
HeLa cells (ATCC, catalog number: CCL-2 ) or HEK293T cells (ATCC, catalog number: CRL-3216 )
pMOS-GFP-Grb2 plasmid (Ketteler et al., 2002)
3xFLAG-TACC3 plasmid (Petschnigg et al., 2017)
Dulbecco modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 61965026 )
Note: Any commercially available DMEM can be used, and GlutaMax can be added, but is not required for HeLa and HEK293T cells.
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500056 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Trypsin/EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: R001100 )
GlutaMax (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
Paraformaldehyde, 4% (v/v) (Santa Cruz Biotechnologies, catalog number: sc-281692 )
Note: Should be stored at -20 °C in aliquots for long-term storage. Thawed aliquots can be kept at 4 °C for up to a month.
Hoechst 33342 trihydrochloride, trihydrate, 1 mg/ml stock (Thermo Fisher Scientific, InvitrogenTM, catalog number: H3570 )
Polyethylenimine (PEI), 10 mg/ml stock in water (Sigma-Aldrich, catalog number: 408727 )
Sodium chloride (NaCl), AnalaR NORMAPUR (VWR, catalog number: 27810.295 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate dibasic (Na2HPO4), AnalaR NORMAPUR (VWR, catalog number: 102494C )
Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P8281 )
PEI solution (see Recipes)
1x phosphate buffered saline (PBS) (pH 7.4) (see Recipes)
10x phosphate buffered saline (PBS) (see Recipes)
Equipment
Multi-channel pipette (Finnpipette, 8-channel P300, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4661030N )
Incubator (Eppendorf, model: Galaxy® 170 R )
High-content screening microscope (PerkinElmer, Opera)
Software
ImageJ
Procedure
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Petschnigg, J. and Ketteler, R. (2017). GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling. Bio-protocol 7(17): e2528. DOI: 10.21769/BioProtoc.2528.
Download Citation in RIS Format
Category
Cancer Biology > Cancer biochemistry > Protein
Cell Biology > Cell staining > Protein
Cell Biology > Cell signaling > Intracellular Signaling
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