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1,591 | https://bio-protocol.org/exchange/protocoldetail?id=1591&type=0 | # Bio-Protocol Content
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Quantification of Respiratory Activity in Biofilms
Cláudia N. H. Marques
SC Scott A. Craver
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1591 Views: 10009
Edited by: Maria Sinetova
Reviewed by: Alexander B. WestbyeEsteban Paredes-Osses
Original Research Article:
The authors used this protocol in Nov 2014
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Abstract
Bacteria live mostly as biofilms, not as planktonic cell populations. Bacterial cells living as biofilms are known to be in different physiological status. Persister cells are one of such physiological conditions and they are recognized as to be a stochastically produced sub-population of non-growing bacterial cells. The following protocol describes a method to determine the respiratory activity of cells within biofilms.
Materials and Reagents
Bacterial culture [Pseudomonas aeruginosa (P. aeruginosa) PA14]. This method has also been performed for Escherichia coli (BW25113)
LB broth Lennox (BD, catalog number: 240230 )
CorningTM cellgroTM Ciprofloxacin Hydrochloride (Thermo Fisher Scientific, catalog number: 61-277-RF )
BacLightTM RedoxSensorTM CTC Vitality Kit (Thermo Fisher Scientific, catalog number: B34956 )
SYTO® 40 Blue Fluorescent Nucleic Acid Stain-5 mM Solution in DMSO (Thermo Fisher Scientific, catalog number: S11351 )
Sodium chloride (VWR International, BDH®, catalog number: 0241-VBD )
Aluminum Foil
Spooled Masterflex peroxide-cured silicone tubing, L/S 14, 250 ft. (Cole-Parmer, catalog number: UX-96407-14 )
Masterflex Norprene tubing (A60 G), L/S 13, 50 ft. (Cole-Parmer, catalog number: UX-06404-13 )
PVDF barbed Y connector, 3/8" ID, 1/4", 2-3/8", 1-3/4"; Pack Of 10 (Cole-Parmer, catalog number: WU-30703-93 )
Barbed fittings, Straight Connector, Clear PP,1/16" ID, 1/32", 25/32", 1/4" (Cole-Parmer, catalog number: WU-30506-00 )
Barbed fittings, Reducing Connector, Clear PP,1/8" x 1/16" ID, 3/32", 13/16", 1/8" (Cole-Parmer, catalog number: WU-30506-06 )
Barbed fittings, T connector, Kynar, 1/4" ID, 1/8", 1-15/16", 1-5/16"; 10/pack (Cole-Parmer, catalog number: WU-30703-75 )
Micropipettes (P1000, P200, P20)
Syringe 10 ml
Syringe needle
Glass microscope slides (25 x 75 x 1 mm)
Glass microscope coverslips (60 x 24 mm; no 2)
Saline (see Recipes)
Equipment
Peristaltic pump (Cole-Parmer, catalog number: EW-07553-80) with an 8 channel, 6 rollers, 3-stop Ismatec minicartridge pump head (Cole-Parmer, catalog number: EW-78002-50 )
Inoculation ports (VWR® Sleeve Stoppers) (VWR International, catalog number: 89097-534 )
GeneMate Incubated Shakers (BioExpress, catalog number: H-2000-M )
Nalgene® Carboys with Handles, Polypropylene, Thermo Scientific (VWR International, catalog number: 16101-084 )
Nalgene® Top WorksTM Aseptic Closure System, Silicone, for Bottles and Carboys, Thermo Scientific (VWR International, catalog number: 2135-8303 )
Acro® 50 Vent Filters, Pall Laboratory (VWR International, catalog number: 28143-616 )
Anodized Transmission flow cell reactors (Biosurface technologies, catalog number: FC 81-Al )
Confocal Scanning Microscope and Analysis software (e. g. Leica Confocal SP5 Imaging system and Software)
Software
COMSTAT software (http://www.comstat.dk)
Intensity Luminance V1 software (http://bingweb.binghamton.edu/~scraver/IL.html)
ImageJ software (http://imagej.nih.gov/ij/)
Leica LAS AF software (Leica)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Marques, C. N. H. and Craver, S. A. (2015). Quantification of Respiratory Activity in Biofilms. Bio-protocol 5(18): e1591. DOI: 10.21769/BioProtoc.1591.
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Category
Microbiology > Microbial biofilm > Biofilm culture
Microbiology > Microbial cell biology > Cell staining
Microbiology > Microbial physiology > Respiration
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1,592 | https://bio-protocol.org/exchange/protocoldetail?id=1592&type=0 | # Bio-Protocol Content
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Peer-reviewed
Transverse Sectioning of Arabidopsis thaliana Leaves Using Resin Embedding
Shweta Kalve*
KS Kumud Saini*
KV Kris Vissenberg
TB Tom Beeckman
GB Gerrit T. S. Beemster
*Contributed equally to this work
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1592 Views: 13122
Edited by: Maria Sinetova
Reviewed by: Moritz BomerRumen Ivanov
Original Research Article:
The authors used this protocol in Dec 2014
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Abstract
The leaf is the major functional part of the shoot performing the bulk of photosynthetic activity. Its development is relatively plastic allowing the plant to adapt to environmental changes by modifying leaf size and anatomy. Moreover, a leaf is made up of various distinct cell layers, each having specialized functions. To understand functional adaptation and the development of the leaf it is essential to obtain cross sections throughout leaf development and at maturity (Kalve et al., 2014). Here, we describe a protocol for transverse sectioning of Arabidopsis thaliana leaves using resin embedding. This protocol provides a reliable platform to yield high quality images of cross sections allowing study of development of various tissue layers across the transversal axis of the leaf. As this method is an adaptation of the protocol developed for the Arabidopsis root tip by Beeckman and Viane (1999) and De Smet et al. (2004), it can easily be modified to accommodate other organs and species.
Keywords: Microscopy Arabidopsis thaliana Leaves Microtomy
Materials and Reagents
Arabidopsis thaliana
Acetic Acid, Glacial (Certified ACS), Fisher Chemical (Fisher Scientific, catalog number: A38-212 )
Formaldehyde solution (Sigma-Aldrich catalog, number: 47629 )
Ethanol (VWR International, catalog number: 83813360 )
Acetic anhydride (Sigma-Aldrich, catalog number: 320102 )
TWEEN® 20 (Sigma-Aldrich, catalog number: P9416 )
NaH2PO4.H2O (Fisher Scientific, catalog number: AC2717500190 )
Technovit 7100 with hardener I and II (Heraeus Kulzer GmbH, catalog number: 64709003 )
Toluidine blue (EMD Millipore Corporation, catalog number: 1159300025 )
DPX mounting medium (a mixture of Distyrene, a Plasticizer and Xylene) (Sigma- Aldrich, catalog number: M1289-10 ML )
Object slides (superfrost plus adhesion slides) (Thermo Fisher Scientific, catalog number: 10143352 )
Glass microtome knives (SPI Easy-cut Ultra/Glass, catalog number: 07668-BA )
Cover slips
Microcentrifuge tubes, PCR tubes or molds (Electron microscopy sciences, catalog number: 70900 )
Phosphate buffer (see Recipes)
Technovit solution (see Recipes)
Technovit solution with hardener-II (see Recipes)
Equipment
Vacuum pump (Welch USA- BRS, catalog number: 2522C-02 )
Vacuum jar
Tweezers
Heated plate (Bekso, model: TH3N )
Rotary microtome (Leica Reichert-Jung, catalog number: 2040 )
Bright field microscope (ZEISS, model: Axio Scope A1 ) fitted with a digital camera (ZEISS, model: AxioCamCm1 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant cell biology > Cell imaging
Plant Science > Plant cell biology > Tissue analysis
Cell Biology > Cell imaging > Fixed-tissue imaging
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1,593 | https://bio-protocol.org/exchange/protocoldetail?id=1593&type=0 | # Bio-Protocol Content
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Peer-reviewed
Morphological Quantification of Nuclei and Mitochondria in Serial Block-face Scanning Electron Microscopy Images
HL Haiyan Lu
NO Nobuhiko Ohno
RR Richard M. Ransohoff
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1593 Views: 8684
Original Research Article:
The authors used this protocol in Jul 2014
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Abstract
Serial Block-face Scanning Electron Microscopy (SBF-SEM or 3D-EM) is a powerful tool to study biological structure in ultrastructural level. Quantification of cellular ultrastructure is useful to providing biological information. This technique requires not only high quality of tissue fixation and ideal sample embedding to preserve structures, but also delicate 3D image scanning and post-processing of images. We have adapted previous method to optimize the EM technique to detect and study cellular ultrastructure. Here we present the method to embed samples for 3D-EM technique and to quantify the morphological parameters of nucleus and mitochondria.
Part I. Tissue embedding for 3D-EM images
Keywords: Electron microscopy Mitochondria Nuclus Morphology
Materials and Reagents
Adult mouse brain / spinal cord
4% Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
Glutaraldehyde solution (GA) (Sigma-Aldrich, catalog number: G5882 )
Sodium Cacodylate Buffer (0.4 M, pH 7.2) (Electronic Microscopy Science, catalog number: 11654 )
Phosphate buffered saline (PBS) (1x Solution, Fisher BioReagents) (Fisher Scientific, catalog number: BP2438-4 )
Potassium ferricyanide (III) (Sigma-Aldrich, catalog number: 702587 )
Thiocarbohydrazide (TCH) (Electronic Microscopy Science, catalog number: 21900 )
Osmium tetroxide 4% solution (Electronic Microscopy Science, catalog number: 19150 )
Uranyl acetate (UA) (Electron Microscopy Sciences, catalog number: 22400 )
Lead Nitrate (Crystalline/Certified ACS), Fisher Chemical (Fisher Scientific, catalog number: L62-100 )
L-aspartic acid solution (Sigma-Aldrich, catalog number: A9256 )
Ethanol, 200 proof (100%), USP, DeconTM Labs (Fisher Scientific, catalog number: 07-678-004 )
Acetone (Electronic Microscopy Science, catalog number: 10010)
EMbed-812 kit (Electronic Microscopy Sciences, catalog number: 14120 )
EMS Molded Flat Embedding Mold (Electronic Microscopy Sciences, catalog number: 70905-01 )
Equipment
Vibratome (Leica, catalog number: Leica VT1000S )
Zeiss Sigma VP SEM (Zeiss) with Gatan 3View (Gatan)
Figure 1. Zeiss Sigma VP SEM (Zeiss) with Gatan 3View (Gatan)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lu, H., Ohno, N. and Ransohoff, R. M. (2015). Morphological Quantification of Nuclei and Mitochondria in Serial Block-face Scanning Electron Microscopy Images. Bio-protocol 5(18): e1593. DOI: 10.21769/BioProtoc.1593.
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Category
Cell Biology > Cell imaging > Fixed-tissue imaging
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1,594 | https://bio-protocol.org/exchange/protocoldetail?id=1594&type=0 | # Bio-Protocol Content
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Peer-reviewed
Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions
Ian Barr
Feng Guo
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1594 Views: 31516
Edited by: Fanglian He
Reviewed by: Seda Ekici
Original Research Article:
The authors used this protocol in Jul 2012
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Jul 2012
Abstract
Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by heme, thus allowing correlation of heme content with activity. This requires the absolute heme concentration and an accurate protein concentration. Another use is to determine the extinction coefficients of a heme-bound protein. This assay is fast, easy, and reproducible if done correctly.
Keywords: heme pyridine hemochromagen hemoprotein porphyrin
Background
G.G. Stokes was the first to prepare what we now refer to as hemochromagen. As early as 1863, he was monitoring changes in the hemoglobin absorbance spectrum upon reduction of the heme to the Fe(II) form Stokes (1863). Stokes had reduced blood in the presence of ammonia; what he was seeing were the intense α and β peaks of Fe(II) heme b from hemoglobin in complex with ammonia. Later authors (Anson and Mirsky, 1928) were able to show that the hemochromagen, as it had been called by Christian Bohr (Edsall, 1972) , was heme in complex with some nitrogenous ligand. It can be formed as well by simply reducing myoglobin under denaturing conditions, in which case histidines serve as the axial ligands. Hill demonstrated that the pyridine hemochromagen is formed from the nitrogen of two pyridine molecules binding to the axial position of the reduced heme (Hill, 1926; Hill, 1929); this was confirmed by Smith (1959) and Gallagher and Elliot (Gallagher et al., 1965; Gallagher et al., 1968).
With advances in spectroscopic techniques, later workers were able to use hemochromagen for an important analytical purpose (Hill, 1929). The regularity of the α peak of reduced pyridine hemochromagen, its high extinction coefficient, and the fact that it follows Beer's Law over a wide range allows its use for determining the total heme composition of a sample. De Duve (1948) published one of the first protocols for this, and determined the extinction coefficient (ε) at 557 nm to be 32 mM-1 cm-1. The method used relied on gravimetric determination of the standard heme samples, which could lead to an underestimate of the true heme concentration if the sample is impure. Paul et al. (1953) re-determined the extinction coefficient for pyridine hemochromogen from recrystallized heme b and myoglobin, using the iron content as an internal control. They found a value (34.7 mM-1 cm-1) significantly higher than the previous value of 32 mM-1 cm-1, representing a difference of roughly 9%. Both values have been in use comparatively recently: e.g. (Scott and Lecomte, 2000; Fushitani and Riggs, 1988; Miyoshi et al., 1997; Yachie et al., 1999; Lee et al., 2000; Huche et al., 2006), which use 32 mM-1 cm-1, and (Sono et al., 1984; Senturia et al., 2012; Sinclair et al., 2001; Berry and Trumpower, 1987), which use 34.7 mM-1 cm-1. In this assay we have taken the value given by Paul et al. (1953) to be most accurate.
[Principle of action] The heme-containing protein solution is first mixed with Solution I (see Recipes) containing pyridine, NaOH and potassium ferricyanide. Pyridine serves as a ligand for heme in the Fe(II) state. NaOH keeps sodium dithionite stable. Potassium ferricyanide ensures that all dissolved heme is in the Fe(III) state at first. Then, excess sodium dithionite in solution is added to reduce the heme from Fe(III) to Fe(II). Another option is to use a pipette tip to add a few grains of solid dithionite to the cuvette, and allow it to dissolve. Recipes similar to the current one have been recommended by Sinclair et al. (2001); Berry and Trumpower (1987); Antonini and Brunori (1971). The concentration of heme in the sample can be determined from the absorbance of the reduced sample; the extinction coefficient for reduced pyridine hemochromagen is 34.7 mM-1 cm-1 at 557 nm for heme b. It can also be done using the difference spectrum between the reduced and oxidized samples. Don't forget, in either case, to take your dilution factor into consideration. This protocol is written for heme b; however, other types of heme form hemochromagens as well and can be quantified using the same technique. See Berry and Trumpower (1987) or Table 1 for extinction coefficients for hemes a and c.
Materials and Reagents
Heme-containing sample
Note: The concentration of heme in your stock solution should be at least 10 μM and not much greater than 80 μM. Dithiothreitol (DTT) and other reducing agents can reduce potassium ferricyanide and may interfere with the oxidized sample, but not with the reduced sample. Any common biological buffer should be compatible with this procedure so long as it has no significant absorbance in the 500 nm to 600 nm range and does not form a complex with heme, as should be the case with all Good's buffers. We have personally used phosphate, tris, HEPES, EPPS, MES, and CHES without issue.
0.5 M NaOH
Pyridine (Sigma-Aldrich, catalog number: 360570 )
Potassium ferricyanide(III) (Sigma-Aldrich, catalog number: 702587 )
Sodium dithionite (Sigma-Aldrich, catalog number: 157953-5 G )
Deionized water
0.2 M NaOH, 40% (v/v) pyridine, 500 μM potassium ferricyanide (see Recipes)
0.1 M potassium ferricyanide (K3[Fe(CN)6]) (see Recipes)
0.5 M sodium dithionite in 0.5 M NaOH (see Recipes)
Equipment
Spectrophotometer with a bandwidth ≤ 2 nm
Fume hood
Quartz or glass cuvette, 1 cm length
Pipettes
Note: It is generally good to have a spectrophotometer capable of relatively low spectral bandwidth (SBW) in order to get highly accurate measurements. It is suggested in the literature that the ratio of SBW to the natural bandwidth (NBW) should be 0.1 or lower in order to get an error of less than 0.5% (Surles and Erickson, 1974; Brodersen, 1954). The α band of reduced pyridine hemochromagen has a NBW of around 20 nm, hence the recommendation of 2 nm or less for SBW. Check with your manufacturer if you are unsure of your SBW. Most modern spectrophotometers have SBW less than 4 nm, corresponding to an error of less than 2%. It is also important to note that this error is not random, but results in an underestimate of the true absorbance; having a higher SBW/NBW ratio leads to a 'flattening' of absorbance peaks. If possible, use a scanning spectrophotometer. This allows you to verify that your spectrum looks similar to the spectrum shown in Figure 1.
Figure 1. Example spectrum of reduced and oxidized pyridine hemochromagen (heme b). The bandwidth is set to 1 nm, with data interval 1 nm.
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Barr, I. and Guo, F. (2015). Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions. Bio-protocol 5(18): e1594. DOI: 10.21769/BioProtoc.1594.
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Category
Biochemistry > Other compound > Heme
Biochemistry > Protein > Interaction
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1,595 | https://bio-protocol.org/exchange/protocoldetail?id=1595&type=0 | # Bio-Protocol Content
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Telomere-mediated Chromosomal Truncation via Agrobacterium tumefaciens or Particle Bombardment to Produce Engineered Minichromosomes in Plants
NG Nathaniel D. Graham*
NS Nathan C. Swyers*
RG Robert T. Gaeta
CZ Changzeng Zhao
JC Jon P. Cody
JB James A. Birchler
*Contributed equally to this work
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1595 Views: 8287
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in May 2008
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Abstract
Minichromosomes are small, autonomously functioning chromosomes that exist separately from the normal chromosomal set. Creation of minichromosomes in plants relies on telomere truncation to remove the chromosome arms and the native telomere sequence and replace them with a transgene together with a new telomere sequence to generate a modifiable small chromosome. Telomere truncation has been accomplished utilizing both Agrobacterium tumefaciens, in which a telomere repeat sequence is cloned into the transformation vector near the right border, and particle bombardment, in which the genes of interest and telomere sequence are co-introduced into the plant. In this protocol we will describe the methods for introducing telomere sequences to both Agrobacterium and gold particles, as well as the methods required to verify that these sequences are intact.
[Introduction] Engineered minichromosomes are autonomously functioning chromosomes that contain all of the necessary components required for maintenance through the cell cycle. The production of engineered minichromosomes has several potential applications for the next generation of genetic engineering (Gaeta et al., 2012). The construction of such chromosomes by assembling a centromere, origin of replication, and selectable marker all capped by telomere sequences, as originally performed in yeast, is not feasible in plants because of the epigenetic nature of centromere sequences (Birchler and Han, 2009; Birchler et al., 2011; Liu et al., 2015). In other words, functional centromeres in plants are determined by chromatin features independent of the underlying DNA and therefore the cloning and re-introduction of centromere sequences will not produce a minichromosome. In contrast to the centromere, the telomere is reliant on sequence, with most plant telomeres containing the same TTTAGGG repeat (Adams et al., 2001). As a result, introduction of telomere sequences during transformation has the potential to confer telomere function.
Because of the epigenetic state of the centromere, engineered minichromosomes in plants need to be produced by cleaving away the chromosome arms from an endogenous centromere that never leaves a cell, a procedure known as the top-down method. This was accomplished with the finding that introduction of the chromosome end sequences, the telomere, would cleave chromosomes at the site of integration (Yu et al., 2006). By including genes of interest in addition to the telomere sequences, the foundation to build engineered minichromosomes to specification was established. This protocol describes the procedure to generate these initial truncated minichromosomes.
The process of creating minichromosomes utilizes standard plant transformation protocols. The only modification is the addition of telomere sequences to the transformation construction so that both a transgene and telomere sequence are introduced into a double stranded break during the transformation process. In some cases, the introduced telomere sequence is recognized by telomere elongation machinery and converted into a functioning telomere. As a result, the acentric fragment distal to the insertion point will be lost, and a minichromosome will be created.
Telomere truncation works well with both Agrobacterium tumefaciens and particle bombardment transformation techniques. Using Agrobacterium, successful minichromosome creation relies on inclusion of telomere sequences in the transformation construct. With particle bombardment, telomere sequences are simply added to the DNA mixture that is adhered to the gold beads before transformation is performed. While the length of telomere sequence required for telomere truncation is not known, a study in Arabidopsis thaliana successfully created truncated chromosomes with telomere repeats as short as 100 bp (Nelson et al., 2011). The study also found, however, that longer telomere sequences were more likely to induce truncation events. As a result, it is suggested that the largest amount of telomere that can be reasonably obtained be used during transformation.
While the concept of including telomere sequences in transformation is relatively simple, working with telomere sequences using current molecular cloning techniques is challenging. The repeated nature of the sequences and the high GC content are inhibitive to polymerase function. As a result, protocols reliant on polymerase function, such as PCR or Sanger sequencing, are not efficient. Additionally, oligonucleotide synthesis technologies are limited for producing telomere repeat sequences at the time of this writing. Traditional cloning utilizing restriction enzymes has been most successful in our work. Isolated telomere sequences, when subjected to agarose gels, do not migrate at the expected sizes, but instead are found as discreet bands or smears throughout the gel, probably because they adopt various secondary structures. Additionally, purification of telomere sequences with gel or column purification is usually inefficient unless the DNA is present in large amounts, making traditional cloning difficult. Adding to these challenges is the observation that long telomere sequences are unstable in microbial cells, and have a tendency to be deleted and shortened over time. As a result, Stbl cells (Invitrogen), which possess the recA1 genotype and are specifically designed to prevent repeated sequences from recombination and thus rearrangement, must be used to maintain the repeat, and multiple clones should be isolated and screened to ensure the full size is present. Additionally, when a clone has been isolated, which contains the desired telomere insert, it is often useful to make a large plasmid extraction that is stored in addition to bacterial stocks.
In order to generate engineered minichromosomes, the protocols presented below were developed. For cloning purposes, the telomere sequence is excised from a gel and ligated to the target plasmid within the agarose mixture. The source of the telomere sequence is plasmid pWY82 (Yu et al., 2006), which contains 2.6 kb of the telomere repeat (TTTAGGG). For particle bombardment, primers are used with a modified PCR program to amplify the telomere repeats, which are gel purified and added to gold particles together with the construct of interest. Whether truncation will be performed with Agrobacterium or particle bombardment, the standard transformation protocol for the species of interest can be followed. Fluorescence in-situ hybridization is then performed to determine if a minichromosome has successfully been produced (Yu et al., 2007).
Keywords: Artificial chromosomes Synthetic chromosomes Telomere truncation Genetic engineering Gene stacking
Materials and Reagents
Target binary plasmid with compatible restriction enzyme cut sites near right border or purified plasmid for co-bombardment
Note: There are many binary vectors available, and any are acceptable for use provided the vector contains the necessary restriction enzyme cut sites to move the telomere fragment from pWY82. A map and sequence of pWY82 can be obtained by contacting the corresponding author. In addition, it is suggested to place the telomere sequences near the right border as in the original truncation plasmid (Yu et al., 2007). It is not known whether placing it inside the left border is effective.
Plasmid pWY82 (Contact corresponding author)
QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
AmbionTM Nuclease-Free Water (not DEPC-Treated) (Fisher Scientific, catalog number: AM9937 )
Restriction Enzymes (New England Biolabs)
Note: Enzymes must be chosen based on compatibility between pWY82 and target vector.
UltraPureTM Low Melting Point Agarose (Life Technologies, InvitrogenTM, catalog number: 16520-050 )
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
Acetic Acid (Sigma-Aldrich, catalog number: 27225 )
Antarctic Phosphatase (New England BioLabs, catalog number: M0289S )
DNA Gel Loading Dye (6x) (Thermo Fisher Scientific, catalog number: R0611 )
GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, catalog number: SM0311 )
Ethidium Bromide (Sigma-Aldrich, catalog number: E7637 )
T4 DNA Ligase (New England BioLabs, catalog number: M0202S )
ElectroMAX Stbl4 Cells (、Thermo Fisher Scientific, catalog number: 11635-018 )
S.O.C Media (Super Optimal Broth with Catabolic repressor) (Thermo Fisher Scientific, catalog number: 15544-034 )
Agar (Sigma-Aldrich, catalog number: A1296 )
Petri Dishes
LongAmp® Taq DNA Polymerase (New England BioLabs, catalog number: M0323S )
Fisher BioReagents LB Broth, Miller (Granulated) (Fisher Scientific, catalog number: BP9723-2 )
Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S4014 )
2x YT medium (Sigma-Aldrich, catalog number: Y2377 )
LB broth (see Recipes)
LB plates (see Recipes)
2x YT broth (see Recipes)
Spectinomycin (see Recipes)
TAE (see Recipes)
Equipment
30 °C incubator
30 °C shaker
4 °C cold room
250 ml baffled culture flasks
Nanodrop spectrophotometer (Thermo Fisher Scientific)
Vacuum concentrator
Gel electrophoresis system
37 °C waterbath
70 °C waterbath
Ultraviolet transilluminator
Electroporator
Thermalcycler
Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, catalog number: A9281 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Graham, N. D., Swyers, N. C., Gaeta, R. T., Zhao, C., Cody, J. P. and Birchler, J. A. (2015). Telomere-mediated Chromosomal Truncation via Agrobacterium tumefaciens or Particle Bombardment to Produce Engineered Minichromosomes in Plants. Bio-protocol 5(18): e1595. DOI: 10.21769/BioProtoc.1595.
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Category
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > DNA modification
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1,596 | https://bio-protocol.org/exchange/protocoldetail?id=1596&type=0 | # Bio-Protocol Content
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Infection Assay of Cyst Nematodes on Arabidopsis Roots
HB Holger Bohlmann
Krzysztof Wieczorek
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1596 Views: 11256
Edited by: Zhaohui Liu
Reviewed by: Daniel F. Caddell
Original Research Article:
The authors used this protocol in Sep 2014
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Abstract
Plant parasitic nematodes are devastating pests on many crops. Juveniles (J2) of cyst nematodes invade the roots to induce a syncytium. This feeding site is their only source of nutrients. Male nematodes leave the roots after the fourth molt to mate with females. The females stay attached to their syncytia throughout their life and produce hundreds of eggs, which are contained in their bodies. When the females die their bodies form the cysts, which protect the eggs. Cysts can survive for many years in the soil until favorable conditions induce hatching of the juveniles.
The beet cyst nematode Heterodera schachtii is a pathogen of sugar beet (Beta vulgaris) but can also complete its life cycle on Arabidopsis roots growing on agar plates under sterile conditions. We present here protocols for a stock culture of H. schachtii and an infection assay on agar plates.
Keywords: Nematode infection assay Cyst nematode Heterodera schachtii Arabidopsis
Materials and Reagents
Heterodera schachtii
Seeds of Sinapis alba cv Albatros
Seeds of Arabidopsis thaliana
Calcium hypochlorite
HgCl2
Tween 20
70% ethanol
GAMBORG B5 VITAMIN MIXTURE (Duchefa Biochemie, catalog number: G0415 )
Daichin agar (Duchefa Biochemie, catalog number: D1004 )
Saccharose
KNO3
MgSO4.7H2O
Ca(NO3)2.4H2O
KH2PO4
FeNaEDTA
H3BO3
MnCl2 or MnCl2.4H2O
CuSO4.5H2O or CuSO4.2H2O
ZnSO4.7H2O
CoCl2.6H2O
H2MoO4 or NaMoO4.2H2O
NaCl
ZnCl2
GELRITETM (Duchefa Biochemie, catalog number: G1101 )
Plastic Petri dishes 9 cm
Plastic Petri dishes 14.5 cm
Aluminum foil
PVC membrane 100 µm mesh (Buddeberg GmbH, catalog number: 9068289 )
PVC membrane 15 µm mesh (Buddeberg GmbH, catalog number: 9068280 )
50 ml syringes
Pipettes
Preparation of water agar plates (see Recipes)
Preparation of Knop medium (see Recipes)
Equipment
Nescofilm (or Parafilm)
Growth chamber at 25 °C with 16 h light/8 h dark
Laboratory glassware
Balance
Stereo microscope
Inverse microscope with camera
Clean bench
Autoclave capable of reaching 120 °C
Axiovert 200M inverse microscope (ZEISS) with an integrated camera (ZEISS, model: AxioCam MRc5 )
Funnel for hatching (Figure 1)
Software
Contour tool of the AxioVision software (ZEISS)
SPSS 12.0 (SPSS Inc.)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bohlmann, H. and Wieczorek, K. (2015). Infection Assay of Cyst Nematodes on Arabidopsis Roots. Bio-protocol 5(18): e1596. DOI: 10.21769/BioProtoc.1596.
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Category
Microbiology > Microbe-host interactions > In vitro model
Microbiology > Microbe-host interactions > Nematode
Plant Science > Plant immunity > Disease bioassay
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1,597 | https://bio-protocol.org/exchange/protocoldetail?id=1597&type=0 | # Bio-Protocol Content
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Detection and Visualization of Specific Gene Transcripts by in situ RT-PCR in Nematode-Infected Arabidopsis Root Tissue
Krzysztof Wieczorek
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1597 Views: 9375
Edited by: Zhaohui Liu
Reviewed by: Daniel F. Caddell
Original Research Article:
The authors used this protocol in Sep 2014
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Abstract
This protocol describes an effective method of in situ RT-PCR that was developed to localize specific gene expression directly in thin cross-sections of nematode feeding sites induced by the cyst nematode Heterodera schachtii (H. schachtii) or the root-knot nematode Meloidogyne incognita (M. incognita) in Arabidopsis roots using DIG (Digoxigenin-11dUTP) labeling coupled with AP (alkaline phosphatase) and nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate-based detection. This method is applicable to any other Arabidopsis root tissue.
Keywords: In situ RT-PCR Gene expression Syncytium Nematode feeding site Heterodera schachtii
Materials and Reagents
Arabidopsis roots
Note: Non-infected root fragments or root fragments containing feeding sites of H. schachtii (syncytia) or M. incognita (giant-cells embedded within galls).
Ethanol
Formaldehyde
Diethylpyrocarbonate-treated water (DEPC-H2O)
Low melting agarose
Petri dishes ( 50 mm)
12/24-well plates
Parafilm
Superglue
RNase Away (Thermo Fisher Scientific-Molecular BioProducts, catalog number: 7005 )
DNase I, RNase-free, HC (50 U/µl) (50 U/ul, 1,000 U) ((Thermo Fisher Scientic, Fermentas, catalog number: EN0523 )
RiboLock RNase Inhibitor (40 U/µl) (RiboLock: 40 U/µl) (Thermo Fisher Scientic, Fermentas, catalog number: EO0381 )
Ethylenediaminetetraacetic acid (EDTA)
SuperScriptTM III Reverse Transcriptase Kit (Life Technologies, InvitrogenTM, catalog number: 18080-093 )
Deoxynucleotides (dNTPs)
Bovine serum albumin (BSA)
DIG (Digoxigenin-11dUTP-alkali stable; 25 nmol/25 µl) (Roche Diagnostics, catalog number: 11093088910 )
BioThermTM Taq DNA Polymerase (GeneCraft, catalog number: GC-002-0100 )
Anti-Digoxigenin-AP, Fab fragments (150 U/200 µl) (Roche Diagnostics, catalog number: 11093274910 )
BCIP®/NBT Liquid Substrate System (Sigma-Aldrich, catalog number: B1911-100 ML )
Fixation solution (see Recipes)
10x phosphate buffered saline (PBS) (see Recipes)
20x SSC (see Recipes)
10x PCR buffer (see Recipes)
10x washing buffer (see Recipes)
Equipment
Vibratome (Leica, model: VT 100 )
Forceps
Heating plate
Laminar flow
PCR cycler
Stereo microscope
Procedure
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How to cite：Wieczorek, K. (2015). Detection and Visualization of Specific Gene Transcripts by in situ RT-PCR in Nematode-Infected Arabidopsis Root Tissue . Bio-protocol 5(18): e1597. DOI: 10.21769/BioProtoc.1597.
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Category
Microbiology > Microbe-host interactions > Nematode
Plant Science > Plant cell biology > Tissue analysis
Cell Biology > Cell staining > Nucleic acid
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1,598 | https://bio-protocol.org/exchange/protocoldetail?id=1598&type=0 | # Bio-Protocol Content
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Pectin Nanostructure Visualization by Atomic Force Microscopy
SP Sara Posé
CP Candelas Paniagua
AK Andrew R Kirby
AG A. Patrick Gunning
VM Victor J Morris
MQ Miguel A Quesada
JM Jose A Mercado
Published: Vol 5, Iss 19, Sep 20, 2015
DOI: 10.21769/BioProtoc.1598 Views: 8934
Edited by: Samik Bhattacharya
Reviewed by: Masahiro Morita
Original Research Article:
The authors used this protocol in Oct 2014
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Abstract
Pectins, complex polysaccharides rich in galacturonic acid, are a major component of primary plant cell walls. These macromolecules regulate cell wall porosity and intercellular adhesion, being important in the control of cell expansion and differentiation through their effect on the rheological properties of the cell wall. In fruits, pectin disassembly during ripening is one the main event leading to textural changes and softening. Changes in pectic polymer size, composition and structure have been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides but studies of detailed structure of isolated polymer chains are scarce (Paniagua et al., 2014). Atomic force microscopy (AFM) is a versatile and powerful technique able to analyze force measurements, as well as to visualize roughness of biological samples at nanoscale (Morris et al., 2010). Using this technique, recent research has found a close relationship between pectin nanostructural complexity and texture and postharvest behavior in several fruits (Liu and Cheng, 2011; Cybulska et al., 2014; Posé et al., 2015). Here, we describe an AFM procedure to topographically visualize pectic polymers from fruit cell wall extracts that has successfully been used in the study of strawberry ripening (Posé et al., 2012; Posé et al., 2015). Thus, from AFM images the 3D structural analysis of isolated chains (length, height, and branch pattern) can be resolved at high magnification and with minimal sample preparation. A full description of AFM fundamentals and the different sampling modes are described in Morris et al. (2010).
Keywords: Pectin Nanostructure AFM Strawberry Cell wall
Materials and Reagents
Tri-distilled butanol (VWR International, catalog number: 20810.323 )
Pectin fractions from cell wall extracts
Notes:
Cell wall extraction protocol is described in Posé et al. (2013).
Pectin fractions from cell wall material are obtained by sequential extractions with CDTA buffer followed by sodium carbonate buffer, to solubilize a cell wall fraction enriched in ionically and covalently bound pectins respectively, as described in Posé et al. (2013) (see Recipes).
Both pectin fractions (i.e., one extracted with CDTA and the other with sodium carbonate) were extensively dialyzed and stored until required at -20 ºC as aqueous solutions. Important: in order to prevent possible aggregation, any freeze-drying step must be avoided. It is recommended to aliquot the samples to avoid freeze-thawed successive cycles.
Ammonium bicarbonate (FLUKA, catalog number: 09830 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 09830”.
Trans-1,2-diaminocyclohexane-N,N,N'N'-tetra-acetic acid monohydrate (Sigma-Aldrich, catalog number: D 319945 )
Sodium carbonate (Sigma-Aldrich, catalog number: S2127 )
NaBH4 (Sigma-Aldrich, catalog number: D 452882 )
10 mM ammonium bicarbonate buffer (pH 8) (see Recipes)
CDTA buffer (see Recipes)
Sodium carbonate buffer (see Recipes)
Equipment
Acoustic-isolated and temperature-controlled room (Figure 1A-1)
Low-power light microscope, equipped with a television camera, was used to roughly position the AFM probe onto the top of the sample (Figure 1A-2)
Atomic Force Microscope (East Coast Scientific Limited, Cambridge, UK) (Figure 1A-3; Figure 2A). Any AFM of suitable resolution can be used, although the details of the software and liquid cell will vary
Anti-vibration table under the AFM microscope (Figure 1A-4)
Photodiode amplifier (Figure 1A-5)
Oscilloscope (Figure 1A-6; Figure 3)
Digital control system composed by computer, DAC (digital-to-analog converter) box, laser driver and high voltage amplifier. (for more details see Morris et al., 2010) (Figure 1A-7)
PYREX® Culture Tubes with Rubber Liner Screw Caps (Thomas Scientific, catalog number: 9212C21 ) and Teflon-lined caps
Note: The screw-capped glass tubes were acid washed using a 1% hydrochloric acid solution overnight then rinsed with water.
Sheets of mica (Elektron Technology, Agar Scientific, model: G250 ) cleaved with adhesive tape (3M, model: Magic Tape ) (Video 1)
Short tip variety AFM probe model contact cantilevers (Budget Sensors SiNi, Bulgaria)
Note: The tip is mounted on the edge of a V-shaped cantilever, the typical geometry used for topographical imaging (Figure 1B).
Tip holder and open bucket liquid cell (Figure 1C; Vdeo 2)
Basic equipment: pipettes, vortex, sonicator bath, heating block
Figure 1. Atomic force microscopy (AFM) equipment. A. Photograph including an overview of AFM room set-up. The numeric labels are in accordance with the equipment list description. B. Scheme of a sharp tip located at the free end of a cantilever. C. Detail of tip holder (left) and liquid cell (right).
Software
AFM software supplied with the instrument (SPM 6.01, ECS, Cambridge, UK)
For the length measurements, images were converted to TIFF files using Paint Shop Pro v5.00 software (http://web.archive.org/web/19980514080113/http://jasc.com/)
Image contrast and 3D effects were optimized using Gwyddion software v2.32
AFM images were analyzed off-line using Image J v1.43u software (http://imagej.nih.gov/ij/index.html)
Gwyddion is free and open source software, covered by GNU (General Public License) (http://gwyddion.net/)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Posé, S., Paniagua, C., Kirby, A. R., Gunning, A. P., Morris, V. J., Quesada, M. A. and Mercado, J. A. (2015). Pectin Nanostructure Visualization by Atomic Force Microscopy. Bio-protocol 5(19): e1598. DOI: 10.21769/BioProtoc.1598.
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Category
Plant Science > Plant biochemistry > Carbohydrate
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Fixed-cell imaging
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1,599 | https://bio-protocol.org/exchange/protocoldetail?id=1599&type=0 | # Bio-Protocol Content
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Physicochemical Quantification of Abscisic Acid Levels in Plant Tissues with an Added Internal Standard by Ultra-Performance Liquid Chromatography
Scott A M McAdam
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1599 Views: 12228
Edited by: Marisa Rosa
Reviewed by: Sara Posé Sriema L. Walawage
Original Research Article:
The authors used this protocol in Apr 2014
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Abstract
The phytohormone abscisic acid (ABA) is critical for a range of plant responses to the environment, most importantly in closing the stomata of seed plants during drought (Mittelheuser and Van Steveninck 1969; Brodribb et al. 2014). The high precision quantification of this hormone by physicochemical methods is a relatively simple process, essential for studies that aim to investigate the role or action of this hormone in plants. Outlined here is a method for the extraction, purification and quantification of ABA levels in plant tissues. This method involves methanolic extraction of ABA from homogenised tissue. Purification of ABA is then undertaken by a simple etheric partitioning method. A focus is placed on determining ABA levels in leaves; however this method is suitable for all tissue types, including plant solutes such as xylem sap, seeds (both dry and green) and large samples of tissue such as root systems.
Keywords: Plant hormones Abscisic acid Stomata Chromatography
Materials and Reagents
Methanol (Merck Schuchardt OHG, catalog number: 603-001-00-X )
2, 6-di-tert-butyl-4-methylphenol (BHT) (Sigma-Aldrich, catalog number: B1378 )
Liquid nitrogen (optional for manual homogenization method only)
Labelled or deuterated ABA (such as 13C labelled, [2H6] ABA or other form with any number of deuterium atoms) (e.g. OlChemIm Ltd, catalog numbers: 03 2721-03 2723 ; catalog numbers: 35671-08-0 )
Acetic acid (Analytical grade, any supplier)
Diethyl ether (Analytical grade, any supplier)
Acetonitrile (for UPLC-MS) (Sigma-Aldrich, catalog number: 271004 )
Aluminium foil
50 ml conical centrifuge tube (Greiner Bio-One GmbH, catalog number: 210261 )
0.5 ml Eppendorf tubes (any brand will suffice)
2 ml screw-cap tubes (Scientific Specialities, catalog number: 2330-00 ) with tethered screw tube caps (Scientific Specialities, catalog number: 2002-59 ) (optional for very small samples or samples that require long-distance freight)
Whatman no. 1 filter paper (optional for large samples only)
1.5 ml Eppendorf tubes (any brand will suffice)
Centrifuge for 2 ml Eppendorf tubes
Filtered glass 150 mm Pasteur pipettes (Poulten & Graf GmbH, model: D810 )
Rubber nipple for Pasteur pipette
10-100 µl pipette (any brand will suffice)
100-1,000 µl pipette (any brand will suffice)
5 ml pipette and tips (any brand will suffice)
80% methanol in water (v/v) (see Recipes)
2% acetic acid (v/v) (see Recipes)
80% methanol in water (v/v) with added butylated hydroxytoluene (BHT) (see Recipes)
80% methanol in water (v/v) (see Recipes)
2% acetic acid (v/v) (see Recipes)
5% methanol, 94% water and 1 % acetic acid (v/v) (see Recipes)
1% acetic acid (v/v) (see Recipes)
Equipment
Balance (±0.001 g)
Scholander pressure chamber (e.g. PMS Instrument Company, model: 600 ) (optional for specialised experiments or quantification of ABA from xylem sap)
Stainless steel beads 7 mm (QIAGEN, catalog number: 69989 / 69990 ) (optional for very small samples only)
Modelling clay or Blu-tak (Bostik) (optional for very small samples only)
Glass beaker (variable size) (optional for large samples)
General purpose 21 cm scissors
Tissue homogeniser [either modified Ba-mix® or Physcotron (Microtec Co., model: NS-7 )]
Cell lysis machine (e.g. QIAGEN, model: TissueLyser II ) (optional for very small samples only)
Conventional Ba-mix® or similar hand-held blender (optional for large samples only)
Ceramic mortar and pestle (optional for manual homogenization method only)
Büchner funnel (optional for large samples only)
Vacuum flask (optional for large samples only)
Vacuum sample concentrator (or rotary evaporator with 100 ml round bottom flask, optional for large samples)
Fume hood
Heating block that can contain 0.5 ml Eppendorf tubes (gentle stream of nitrogen gas, passing through a low flow regulator, tubing and funnelled through a standard 100 µl pipette tip, if desired) (e.g. Ratek Instruments, model: DBH10DP )
A LCGC Certified Clear Glass 12 x 32 mm Screw Neck Vial, with Cap and PTFE/silicone Septum , 2 ml Volume, 100 /pkg [(Waters, catalog number: 186000272C ), Insert 150 µl with preinstalled plastic spring (Waters, catalog number: WAT094171 )]
Aerosol barrier pipette tips for both µl pipettes (these are important to prevent sample contamination of the pipette which can compromise future samples. They can be made by inserting a small amount of cotton wool into the barrel end of the pipette tip.) (any brand will suffice)
Ultra-performance liquid chromatograph and multiple reaction-monitoring tandem mass spectrometer [Water Acquity H-series UPLC coupled to a Waters Xervo triple quadrupole mass spectrometer, containing a Waters Acquity UPLC BEH C18 column (2.1 mm x 100 mm x 1.7 µm particles)]
Software
Waters MassLynx and TargetLynx software
Procedure
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Copyright: © 2015 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:
McAdam, S. A. M. (2015). Physicochemical Quantification of Abscisic Acid Levels in Plant Tissues with an Added Internal Standard by Ultra-Performance Liquid Chromatography. Bio-protocol 5(18): e1599. DOI: 10.21769/BioProtoc.1599.
McAdam, S. A. and Brodribb, T. J. (2014). Separating active and passive influences on stomatal control of transpiration. Plant Physiol 164(4): 1578-1586.
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Category
Plant Science > Plant biochemistry > Plant hormone
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16 | https://bio-protocol.org/exchange/protocoldetail?id=16&type=0 | # Bio-Protocol Content
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Spot Assay for Yeast
Bio-protocol Editor
Published: Vol 2, Iss 1, Jan 5, 2012
DOI: 10.21769/BioProtoc.16 Views: 41928
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Abstract
This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies.
Materials and Reagents
Yeast cells
YES medium
Equipment
Multichannel Pipetman (Eppendorf)
OmniTray (V&P Scientific)
Microfuge tube
Standard laboratory spectrophotometer
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial cell biology > Cell viability
Microbiology > Microbial cell biology > Cell isolation and culture
Cell Biology > Cell isolation and culture > Cell growth
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160 | https://bio-protocol.org/exchange/protocoldetail?id=160&type=0 | # Bio-Protocol Content
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Peer-reviewed
Calcium Mobilisation Assay in Response to Chemokine Stimulation
DC Darran G. Cronshaw
Published: Vol 2, Iss 9, May 5, 2012
DOI: 10.21769/BioProtoc.160 Views: 14869
Original Research Article:
The authors used this protocol in Jun 2006
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Abstract
This assay is used to measure calcium mobilization in lymphocytes (either in primary cells or cell lines) in response to chemokine stimulation using ratiometric analysis. It has also been used for measuring TCR mediated calcium flux. In addition, the same labeling procedure with the addition of brilliant black (a quenching agent) (Sigma-Aldrich, catalog number: 211842) in the loading buffer (at 100 μM) allows for quantification using the FLIPR system on poly-D-lysine plates. Probenicid is an anion-exchange protein inhibitor and prevents the extrusion of the dyes by organic ion transporters.
Materials and Reagents
Cells of choice
RPMI 1640 media (Life Technologies, Invitrogen™, catalog number: 11875-119 )
Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 10437-028 )
PTX (List Biological Labs, catalog number: 183 )
HBSS (Life Technologies, Invitrogen™, catalog number: 14185-052 )
BSA (Sigma Aldrich, catalog number: A7030 )
HEPES (Life Technologies, Invitrogen™, catalog number: 15630-080 )
Probenecid (Sigma Aldrich, catalog number: P8761 )
Fluo-4 (Life Technologies, Invitrogen™, catalog number: F14201 )
Fura-red (Life Technologies, Invitrogen™, catalog number: F3021 )
Pluronic F-127 (Life Technologies, Invitrogen™, catalog number: P-3000MP )
DMSO (Sigma Aldrich, catalog number: D2650 )
Ionomycin (Sigma Aldrich, catalog number: I0634 )
NaOH (Thermo Fisher Scientific, catalog number: BP359-212 )
Assay buffer (see Recipes)
Loading buffer (see Recipes)
Equipment
FCM (FACS LSR II machine)
FACS tube
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Cronshaw, D. G. (2012). Calcium Mobilisation Assay in Response to Chemokine Stimulation. Bio-protocol 2(9): e160. DOI: 10.21769/BioProtoc.160.
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Category
Immunology > Immune cell function > Lymphocyte
Cell Biology > Cell-based analysis > Ion analysis
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1,600 | https://bio-protocol.org/exchange/protocoldetail?id=1600&type=0 | # Bio-Protocol Content
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Detection of the Cell Proliferation Zone in Leaves by Using EdU
Hokuto Nakayama
KK Kensuke Kawade
Hirokazu Tsukaya
Seisuke Kimura
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1600 Views: 8485
Edited by: Tie Liu
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Dec 2014
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Dec 2014
Abstract
Visualization of nuclei in S-phase cells in tissues is important for not only cell cycle research but also developmental research because morphogenesis is usually achieved by a combination of cell proliferation and cell expansion. Recently, DNA labeling with 5-ethynyl-2′-deoxyuridine (EdU), which is an analog of thymidine, has been used to visualize nuclei in S-phase cells to assess the activity of cell proliferation during development of plants. EdU is efficiently incorporated into newly synthesized DNA, and detection of EdU is based on the covalent reaction between EdU and Alexa Fluor® dye, which is one of useful fluorescent dyes; this allows us to use mild conditions for the assay without any DNA denaturation. This method could be easily applicable, and, indeed, has been used for various model and non-model plant species. Here, we have described a protocol developed for the detection of nuclei in S-phase cells in leaves.
Keywords: Cell division Cell proliferation 5-ethynyl-2′-deoxyuridine (EdU) Rorippa aquatica
Materials and Reagents
Plant tissues
Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: c10337 )
90 ml acetone (WAKO, catalog number: 019-00353 )
1x phosphate-buffered saline (PBS) (e.g. WAKO, catalog number: 314-90185 )
0.5% Triton X-100 in 1x PBS
Plastic tubes (PCR tube or 1.5-ml tube)
Aluminum foil
90% acetone (see Recipes)
Fixative formalin-acetic acid-alcohol (FAA) (see Recipes)
10 mM EdU stock solution (see Recipes)
10 µM EdU solution (see Recipes)
Alexa Fluor® 488 azide solution (see Recipes)
Click-iT® EdU reaction buffer (see Recipes)
Click-iT® EdU buffer additive (see Recipes)
Reaction cocktail (see Recipes)
Equipment
Microscope (e.g. Nikon, model: ECLIPSE 80i )
Shaker
Vacuum pump
Desiccator
Razor
Tweezer
Procedure
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How to cite：Nakayama, H., Kawade, K., Tsukaya, H. and Kimura, S. (2015). Detection of the Cell Proliferation Zone in Leaves by Using EdU. Bio-protocol 5(18): e1600. DOI: 10.21769/BioProtoc.1600.
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Category
Plant Science > Plant developmental biology > Morphogenesis
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > DNA labeling
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1,601 | https://bio-protocol.org/exchange/protocoldetail?id=1601&type=0 | # Bio-Protocol Content
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Isolation and Growth of Adult Mouse Dorsal Root Ganglia Neurons
Seong-il Lee
JL Joel Levine
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1601 Views: 26232
Edited by: Xuecai Ge
Reviewed by: Hong-guang XiaAntoine de Morree
Original Research Article:
The authors used this protocol in Feb 2013
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Feb 2013
Abstract
Adult dorsal root ganglia neurons are among the few adult neuronal cell types that can be purified and grown relatively easily in dissociated cell culture. Here we describe a procedure for the isolation and growth of dissociated adult mouse DRG neurons using Percoll gradients and a chemically defined medium. These cultures can be used for morphological, biochemical and electrophysiological studies of neuronal growth and function.
Keywords: Primary culture Dorsal root ganglia Neuron Percoll gradient Rodent
Materials and Reagents
6 to 9 weeks old female mouse
Poly-L-lysine hydrobromide (100 μg/ml in water) (Sigma-Aldrich, catalog number: P1524 )
Laminin (1 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 23017-015 )
Collagenase from Clostridium histolyticum (10 mg/ml in Ca2+, Mg2+ free PBS) (Sigma-Aldrich, catalog number: C-9891 )
Dispase II (10 mg/ml in Ca2+, Mg2+ free PBS) (Roche Diagnostics, catalog number: 04-942-078-001 )
Deoxyribonuclease I from bovine pancreas (4 mg/ml in PBS) (Sigma-Aldrich, catalog number: DN25 )
Leibovitz's L-15 Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11415-064 )
Neurobasal®-A Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888-022 )
B-27® supplement (50X), serum free (Thermo Fisher Scientific, InvitrogenTM, catalog number: 17504-044 )
Percoll® (Sigma-Aldrich, catalog number: P1644 )
Hank’s Balanced Salt Solution (HBSS, calcium, magnesium, no phenol red) (Thermo Fisher Scientific, GibcoTM, catalog number: 1425092 )
15 mm Glass coverslips (Neuvitro, catalog number: GG-15 )
Pasteur pipette
Falcon® 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish, 20/Pack, 500/Case, Sterile (Corning Incorporated, Falcon®, catalog number: 351008 )
Falcon® 60 mm x 15 mm Not TC-Treated Treated Bacteriological Petri Dish, 20/Pack, 500/Case (Corning Incorporated, Falcon®, catalog number: 351007 )
Falcon® 14 ml Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap, Sterile, 25/Pack, 500/Case (Corning Incorporated, Falcon®, catalog number: 352059 )
Falcon® 14 ml Round Bottom PP Test Tube, without Cap, Sterile, 125/Pack, 1000/Case (Corning Incorporated, Falcon®, catalog number: 352018 )
Equipment
#10 scalpel blade (Fine Science Tool, catalog number: 10010-00 ) (Figure 1)
Standard scalpel handle #3 (Fine Science Tool, catalog number: 10003-12 ) (Figure 1)
Spring Scissors-Angled to Side (Fine Science Tool, catalog number: 15006-09 ) (Figure 1)
Small curved scissors (Fine Science Tool, catalog number: 14095-11 ) (Figure 1)
Standard Pattern Forceps (Fine Science Tool, catalog number: 11000-12 ) (Figure 1)
Dumont #5 forceps (Fine Science Tool, catalog number: 11252-20 ) (Figure 1)
Dissecting microscope
Swinging bucket rotor (Sorvall instrument, model: HB-4 )
Centrifuge (Sorvall instrument, model: RC5 plus )
Figure 1. The dissection tools for mouse dissection and DRG isolation. A. Standard scalpel handle #3; B. Spring scissors; C. Small curved scissors; D. Standard forceps; E. Dumont #5 forceps.
Procedure
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Copyright: © 2015 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:
Lee, S. and Levine, J. (2015). Isolation and Growth of Adult Mouse Dorsal Root Ganglia Neurons. Bio-protocol 5(18): e1601. DOI: 10.21769/BioProtoc.1601.
Lee, S. I., Zhang, W., Ravi, M., Weschenfelder, M., Bastmeyer, M. and Levine, J. M. (2013). Atypical protein kinase C and Par3 are required for proteoglycan-induced axon growth inhibition. J Neurosci 33(6): 2541-2554.
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Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Cell isolation and culture > Cell growth
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1,602 | https://bio-protocol.org/exchange/protocoldetail?id=1602&type=0 | # Bio-Protocol Content
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Peer-reviewed
Measurement of Resting Energy Metabolism in Mice Using Oxymax Open Circuit Indirect Calorimeter
Yaohui Nie
TG Timothy P. Gavin
SK Shihuan Kuang
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1602 Views: 10517
Reviewed by: Xuecai Ge
Original Research Article:
The authors used this protocol in Aug 2014
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Abstract
Indirect calorimeter is a powerful tool to monitor resting energy metabolism through the measurement of oxygen (O2) consumption and carbon dioxide (CO2) production. From the measurement of VO2 and VCO2, the respiratory exchange ratio (RER) can be calculated to assess energy fuel utilization and energy expenditure (Evan et al., 2012). Previously, indirect calorimeter has been widely used in metabolic disease research in mice to reveal the potential roles of specific genes or treatments in regulating energy metabolism (for example: Bi et al., 2014; Feng et al., 2014). Here, we described a protocol to evaluate the resting energy metabolism of C57BL/6 mice during dark and light cycles using the Oxymax Open Circuit indirect calorimeter.
Keywords: Brown adipose tissue Skeletal muscle Oxygen consumption Insulin resistance Diabetes
Materials and Reagents
Adult mice (C57BL/6 male mice at 3-month old were used for data acquisition in this protocol, but male or female mice of other genetic backgrounds or strains, at different ages can be used)
Food (normal chow diet or high fat diet) and water (ad lib)
Compressed gas mixture with the components of 4,929 PPM CO2, 20.47% O2 and Balance N2
Equipment
Oxymax Open Circuit Indirect Calorimeter (Columbus Instruments, model: Open Circuit Indirect Calorimeter) (Figure 1)
Figure 1. Open circuit indirect calorimeter components
Computer with software provided by the manufacture (Columbus Instruments, model: Oxymax v4.91 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Nie, Y., Gavin, T. P. and Kuang, S. (2015). Measurement of Resting Energy Metabolism in Mice Using Oxymax Open Circuit Indirect Calorimeter. Bio-protocol 5(18): e1602. DOI: 10.21769/BioProtoc.1602.
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Category
Cell Biology > Cell metabolism > Other compound
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1,603 | https://bio-protocol.org/exchange/protocoldetail?id=1603&type=0 | # Bio-Protocol Content
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5’ Rapid Amplification of cDNA Ends (5’ RACE) of Agrobacterial T-DNA Genes within Transformed Plant Sample
YZ Yi Zhang
Published: Vol 5, Iss 18, Sep 20, 2015
DOI: 10.21769/BioProtoc.1603 Views: 11840
Edited by: Arsalan Daudi
Reviewed by: Kanika Gera
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
The T-DNA (transferred-DNA) region of virulent Agrobacterium tumefaciens (A. tumefaciens) strain is transferred and integrated into the plant genome, and thereby the T-DNA genes are expressed in transformed plant cells. This protocol was used to analyze the transcription start sites (TSSs) of agrobacterial T-DNA genes within plant crown gall tumor. Firstly, the stems of Arabidopsis thaliana were inoculated by A. tumefaciens strain C58 and developed crown gall tumor. Subsequently, the mRNA was extracted from the crown gall tumor and then used for amplification of 5’ cDNA ends by 5’ Rapid Amplification of cDNA Ends (5’ RACE) assay. The full-length cDNAs were generated in reverse transcription reactions and used to analyze TSSs. Here, TSSs of three oncogenes, IaaH, IaaM and Ipt were analyzed as examples. This protocol also allows for identification of TSSs of the other agrobacterial T-DNA genes that expressed in plant cells.
Keywords: Agrobacterium tumefaciens T-DNA 5' RACE Transcription start site
Materials and Reagents
A. tumefaciens strain C58 nocc (nopaline catabolism) (MAX-PLANCK-GESELLSCHAFT, catalog number: 584 )
Arabidopsis thaliana Col-0
Escherichia coli (E. coli) strain MRF’ (Agilent Technologies, catalog number: 200230-41 )
Dynabeads® Oligo(dT)25 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 61005 )
SMARTer® RACE 5’/3’ Kit (Takara Bio Company, Clontech, catalog number: 634859 )
DreamTaq DNA Polymerase (5 U/µl) (Thermo Fisher Scientific, catalog number: EP0701 )
QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
pGEM®-T Easy Vector Systems (Promega Corporation, catalog number: A1360 )
Hypodermic Syringes without Needle (5 cc) (Terumo Medical Corporation, catalog number: SS-05L )
Hypodermic Needles (Terumo Medical Corporation, catalog number: NN-2138R )
KB medium (see Recipes)
Agromix buffer (see Recipes)
Lysis buffer (see Recipes)
Washing buffer A (see Recipes)
Washing buffer B (see Recipes)
Elution buffer (see Recipes)
2x binding buffer (see Recipes)
Equipment
Culture tubes 13 ml (SARSTEDT, catalog number: 62.515.028 )
NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Fisher Scientific, catalog number: ND-2000c )
High-performance, Modular Stereomicroscope for Application Based Customization MZ6 (Leica Microsystems, model: MZ6)
Ball mill (RETSCH, model: MM200 )
Thermal cycler (Eppendorf, catalog number: 950000031 )
MiniSpin®/MiniSpin® plus Centrifuge (Eppendorf, catalog number: 0 22620100 )
MagneSphere® Technology Magnetic Separation Stands (Promega Corporation, catalog number: Z5342 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant molecular biology > DNA
Plant Science > Plant transformation > Agrobacterium
Molecular Biology > DNA > PCR
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1,604 | https://bio-protocol.org/exchange/protocoldetail?id=1604&type=0 | # Bio-Protocol Content
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3’ Rapid Amplification of cDNA Ends (3’ RACE) Using Arabidopsis Samples
ER Encarnación Rodríguez-Cazorla
Alfonso Andújar
Juan José Ripoll
LB Lindsay J. Bailey
Antonio Martínez-Laborda
Martin F. Yanofsky
Antonio Vera
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1604 Views: 19823
Edited by: Arsalan Daudi
Reviewed by: Fang Xu
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
Production of functional eukaryotic RNA is a very elaborate process that involves a complex interplay between transcription and various RNA processing activities, including splicing, 5’ capping, and 3’ cleavage and polyadenylation (Bentley, 2014). Accurate mapping of RNA ends provides a valuable tool to assess transcriptional and post-transcriptional events giving rise to different gene transcripts. The abundance of such transcripts most likely depends on exogenous and developmental cues, or mutations. In the reference plant Arabidopsis, perturbation of the HUA-PEP post-transcriptional regulatory factors (Rodríguez-Cazorla et al., 2015) leads to the accumulation of aberrant transcripts of the key floral homeotic gene AGAMOUS (AG) (Yanofsky et al., 1990) that retain intronic sequence. It was determined by 3’ RACE reactions that such erroneous transcripts correspond to premature processing and polyadenylation events taking place at the AG intron region. Here we describe a protocol that is suitable for analysis of relatively abundant transcripts and also for detecting aberrant RNA species that are likely prone to rapid turnover. Likewise, the method, here adapted to Arabidopsis reproductive tissues, can be applied to characterize RNA species from other organs (leaf, root) and/or other plant species. We provide a detailed protocol of our 3’ RACE procedure comprising four major parts: Total RNA extraction, RNA amount determination and quality control, the RACE procedure itself, and isolation of the resulting RACE products for cloning and sequencing.
Keywords: Arabidopsis 3' RACE RNA extraction
Materials and Reagents
Disposable gloves
Sterile disposable RNase-free pipette tips
RNase-free microcentrifuge tubes
Plant sample (Arabidopsis young flower buds, stages 1 through 9)
Note: Arabidopsis flower stages according to Smyth et al., 1990. Other tissue/species can be tested as well.
Liquid nitrogen
GeneJET Plant RNA Purification Kit (Thermo Fisher Scientific, catalog number: K0801 )
1 M DTT (Sigma-Aldrich, catalog number: 43816 )
Absolute ethanol (JT Baker 8006) and 96% Ethanol (as recommended by the RNA extraction kit manufacturer, see point 6 above)
4M LiCl (made in distilled water and autoclaved, not necessarily fresh)
DNase I, RNase-free (Thermo Fisher Scientific, catalog number: EN0525 )
RiboLock RNase Inhibitor (Thermo Fisher Scientific, catalog number: EO0382 )
dNTPs mix 10 mM each (Thermo Fisher Scientific, catalog number: R0192 )
OligodT-Anchor Primer (Roche 5’/3’ RACE Kit) (Roche Diagnostics, catalog number: 03 353 621 001 , version 10)
Maxima Reverse Transcriptase + buffer 5x (Thermo Fisher Scientific, catalog number: EP0741 )
RNase-free water
High Fidelity PCR Enzyme Mix + buffer 10x (Thermo Fisher Scientific, catalog number: K0191 )
PCR Anchor Primer (Roche 5’/3’ RACE Kit) (Roche Diagnostics, catalog number: 03 353 621 001 )
Gene specific 5’ Primer/s (Table 1)
GeneJET Gel Extraction Kit (Thermo Fisher Scientific, catalog number: K0691 )
StrataClone PCR Cloning Kit (Agilent Technologies, catalog number: 240205 )
Taq DNA Polymerase (EURx, catalog number: EK2500-04 )
GeneRulerTM 100 bp Plus DNA Ladder (Thermo Fisher Scientific, catalog number: SM0321 ) (or any other suitable molecular marker for your convenience)
Equipment
Mortar and pestle (beaked Haldenwanger mortar, 63 mm. diameter. Clean, autoclave and dry before use)
Thermomixer (water bath or heat block can be used as well) (Eppendorf)
Microcentrifuge (Heraeus, Biofuge Pico)
Refrigerated centrifuge BR15 (B. Braun Melsungen AG) with rotor 12,148-H (Sigma-Aldrich) for 1.5 ml Eppendorf tubes
BioPhotometer Plus (Eppendorf)
Thermal Cycler T100 (Bio Rad) (or any other conventional PCR device)
Electrophoresis system
UV transilluminator
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Rodríguez-Cazorla, E., Andújar, A., Ripoll, J. J., Bailey, L. J., Martínez-Laborda, A., Yanofsky, M. F. and Vera, A. (2015). 3’ Rapid Amplification of cDNA Ends (3’ RACE) Using Arabidopsis Samples. Bio-protocol 5(19): e1604. DOI: 10.21769/BioProtoc.1604.
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Category
Plant Science > Plant molecular biology > DNA
Plant Science > Plant molecular biology > RNA
Molecular Biology > DNA > PCR
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1,605 | https://bio-protocol.org/exchange/protocoldetail?id=1605&type=0 | # Bio-Protocol Content
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Peer-reviewed
Pot Level Drought Stress Tolerance Assay in Tobacco through Plant Phenotyping and Antioxidant Assay
Priyanka Das
NL Nita Lakra
KN Kamlesh Kant Nutan
Sneh L Singla-Pareek
Ashwani Pareek
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1605 Views: 12182
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Oct 2013
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Oct 2013
Abstract
Drought is an important abiotic factor which has a huge detrimental impact on crop productivity. Study of plant responses towards drought stress and investigating the mechanism of drought tolerance is crucial for achieving the target of developing drought-tolerant plants. Phenotyping is a cost effective approach which can be adopted to evaluate the severity of drought stress in a plant. Next to phenotyping parameters, biochemical parameters such as the study of antioxidant enzyme activity play significant roles in assessing the extent of drought stress caused injury in a plant. Among the antioxidant enzymes, ascorbate peroxidase is an enzyme which plays a crucial role in drought tolerance in plants. It has been well established that the activity of this enzyme increases under drought stress. Here, we present a simple and reproducible protocol to investigate the response of tobacco plants towards drought stress through measurement of phenotypic parameters and antioxidant enzyme activity. Though, these experiments have been conducted with tobacco plants, this protocol could be adopted for other crop species.
Keywords: Drought Tolerance Antioxidant Ascorbate peroxidase Phenotyping
Materials and Reagents
Pipette (BrandTech Scientific, Transferpette, catalog numbers: 704770 , 704778 , 704780 )
Centrifuge tube (1.5 ml) (Corning, Axygen®, catalog number: MCT-150-C-S )
Tobacco seed samples under study: Nicotiana tabacum L. cv. Petit Havana, wild type and transgenic lines
Note: The complete open reading frame of histone-gene binding protein of rice (OsHBP1b) was cloned at BglII and SpeI sites in the plant expression vector (pCAMBIA1304) and used for transformation of Agrobacterium strain (LBA 4404). Fifteen days old tobacco seedlings were used for transformation through tissue culture method. The putative transgenic T0 plants were transferred to the greenhouse for the purpose of seeds multiplication. The seeds of T2 transgenic lines were used for experimental purpose.
Distilled H2O (IndiaMART InterMESH Ltd, Mars Scientific Instruments Co., catalog number: BASIC/pH & XL )
Agro peat (Agro peat super) (AswiniAgrotech)
Vermiculite (Manidharma Biotech Private Limited)
Sucrose (Sigma-Aldrich, catalog number: S0389 )
Stress reagent (Polyethylene glycol 6000 or PEG 6000) (Sigma-Aldrich, catalog number: 81260 )
Ascorbate (Sigma-Aldrich, catalog number: A4034 )
Liquid Nitrogen (Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi)
Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: 216763 )
Bovine serum albumin (Sigma-Aldrich, catalog number: A2153 )
Murashige & Skoog (MS) with vitamins (Caisson Laboratories, catalog number: MSP09-1lt )
K2HPO4 (Sigma-Aldrich, catalog number: 1551128 )
KH2PO4 (Sigma-Aldrich, catalog number: 1551139 )
Coomassie Brilliant Blue G-250-100 mg (Sigma-Aldrich, catalog number: 27815 )
Methanol (100%) (Sigma-Aldrich, catalog number: 34860 )
Phosphoric acid (85%) (Sigma-Aldrich, catalog number: W290017 )
Phosphate buffer (see Recipes)
Bradford reagent (see Recipes)
MS media (see Recipes)
Equipment
Plastic pots (12 cm diameter and 13 cm depth) (Garden Aids, India)
Forceps (ACE Surgical Co, catalog number: 10)
Growth chamber (Daihan LabTech India Pvt. Ltd., model: LGC-S201 )
Oven (Hicon India)
pH meter (Control Dynamic Instrumentation Pvt. Ltd., model: APX175E )
Note: Currently, it is “CD Hightech Pvt. Ltd., model: APX175E ”.
Plastic ice tray
Liquid nitrogen container (Kailash Gases, Cryocan, model: BA-11 )
Motor and pestle (local supplier, New Delhi)
Cuvette (both plastic and quartz) (Sigma-Aldrich, catalog number: Z276677 )
Spectrophotometer (Cary 300 UV-Vis)
Image capturing device (SONY CORPORATION OF AMERICA, model: DSC-HX300 )
Weighing machine (Sartorius, model: BSA224S-CW )
Scale (30 cm plastic scale)
Software
GraphPad InStat3 software
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant physiology > Abiotic stress
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1,606 | https://bio-protocol.org/exchange/protocoldetail?id=1606&type=0 | # Bio-Protocol Content
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Peer-reviewed
TCRβ Clonotype Analysis of EBV and CMV-specific Human CD8+ T Cells
NN Nening M. Nanlohy
DK Dan Koning
EQ Esther D. Quakkelaar
DB Debbie van Baarle
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1606 Views: 7674
Reviewed by: Kathrin SutterMarielle Cavrois
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
This protocol describes the quantification of all expressed T-cell antigen receptor (TCR) gene products within sorted (by flow cytometry) EBV and CMV-specific memory CD8+ T-cell populations using a template-switch anchored reverse transcription polymerase chain reaction (RT-PCR).
Materials and Reagents
Nuclease-free 1.5 ml tubes (Sarstedt, catalog number: 72692005 )
RNAlater (Life Technologies, catalog number: AM7020 )
Note: Currently, it is “Thermo Fisher Scientific, AmbionTM, catalog number: AM7020”.
FACS-sorted CD8+ T cells in RNAlater (Koning et al., 2013)
T-cell markers CD3 (PerCP) (BD), CD8 (AmCyan/V500) (BD) and pMHC-tetramer (APC/PE) (home-made), and the dump markers CD14 and CD19 (both BioLegend, Pacific Blue®)
LIVE/DEAD Viability dye (Thermo Fisher Scientific, InvitrogenTM)
µMACS mRNA isolation kit (Miltenyi Biotec, catalog number: 130-075-201 )
SMARTer Pico PCR cDNA synthesis kit (Takara Bio Company, Clontech, catalog number: 634928 )
dNTPs 25 mM each (Life Technologies, catalog number: R1121 )
Recombinant RNasin Ribonuclease Inhibitor 20 U/μl (Promega Corporation, catalog number: N2511 )
Superscript II RNase H- Reverse transcriptase 200 U/μl (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
Note: This kit also contains dithiothreitol (DTT, 100 mM).
Nucleospin Extract II, gel and PCR clean up (MACHEREY-NAGEL GmbH & Co, catalog number: MN740609.250 )
Molecular grade H2O (Sigma-Aldrich, catalog number: W4502-1L )
Advantage 2 Polymerase Mix (Takara Bio Company, Clontech, catalog number: 639201 )
Molecular grade agarose (Thermo Fisher Scientific, Fisher BioReagents®, catalog number: 10688973 )
SYBR® Gold Nucleic Acid Gel Stain (Life Technologies, catalog number: S-11494 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: S-11494”.
1 kb Plus DNA ladder (Life Technologies, catalog number: 10787-018 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10787-018 ”.
pGEM-T Easy Vector System kit I (Promega Corporation, catalog number: A1360 )
Chemically competent E. coli DH5α (Home-made)
AmpliTaq DNA Polymerase with buffer II (Life Technologies, catalog number: N8080153 )
Note: Currently, it is “Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8080153”.
Big Dye Terminator v3.1 cycle kit (Life Technologies, catalog number: 4337455 )
Note: Currently, it is “Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4337455”.
RNase-inactivator (RNase Away) (MP Biomedicals)
Primers (can be ordered from any company that provides custom made DNA oligo’s):
SMART II oligo
5’-AAGCAGTGGTATCAACGCAGAGTACGCGGG*-3’
Universal
Primer Long
5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’
Universal
Primer Short
5’-CTAATACGACTCACTATAGGGC-3’
MBC2
5’-TGCTTCTGATGGCTCAAACACAGCGACCT-3’
M13 Forward
5’-TTTTCCCAGTCACGAC-3’
M13 Reverse
5’-CAGGAAACAGCTATGAC-3’
Oligo d(T)25
5’-d(T)25VN**-3’
*The final GGG bases in this oligo are RNA bases
**V = A, C, or G. N = A, C, G, or T
Note: Preferably, order primers with high purity (HPLC purified).
10x Universal Primer mix (see Recipes)
50 mM DTT (see Recipes)
Equipment
FACSAria III flow cytometer (BD)
Heating blocks (42 °C and 70 °C) (VWR International)
T-M-AR, DNA/RNA UV-cleaner box (Laminar flow hood/ UVC) (Biosan, catalog number: BS-040104-A06 )
Standard table top Eppendorf centrifuge (for use at room temperature and 4 °C)
PCR thermo cycler
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Immune cell function > Lymphocyte
Microbiology > Microbe-host interactions > In vivo model
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1,607 | https://bio-protocol.org/exchange/protocoldetail?id=1607&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro Studies: Inhibition of Nevirapine Metabolism by Nortriptyline in Hepatic Microsomes
Iris Usach
José-Esteban Peris
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1607 Views: 7697
Reviewed by: Vamseedhar Rayaprolu
Original Research Article:
The authors used this protocol in Dec 2014
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Dec 2014
Abstract
One of the most prevalent and interfering psychosocial comorbidities of HIV infection is clinical depression (22 to 45%). For this reason, a study of a possible interaction between the nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) and the tricyclic antidepressant nortriptyline (NT) was carried out. In vitro studies with rat and human hepatic microsomes showed a marked inhibition of NVP metabolism by NT being more intense in rat than in human. The extrapolation of these results to humans suggests increased NVP side effects when both drugs are coadministered, but additional in vivo human studies are required to evaluate the clinical implication of this interaction.
This protocol describes a technique for detecting and measuring the inhibition of the nevirapine metabolism by nortriptyline in hepatic microsomes.
Keywords: Nevirapine Nortriptyline Metabolism inhibition Drug interaction Hepatic microsomes
Materials and Reagents
10 ml polypropylene tube 16 x 95 mm (Deltalab, catalog number: 400900 )
5 ml ultracentrifuge tube 13 x 51 mm (Beckman Coulter, catalog number: 326819 )
1.5 ml screw cap vial 32 x 11.6 mm
7.5 ml screw cap tube 13 x 100 mm
Wistar rat livers
Saline solution (Laboratorios ERN, catalog number: 999790 )
Protein Standard (2 mg BSA) (BD Biosciences, Falcon®, catalog number: P5619 )
Note: Currently, it is “Sigma-Aldrich, catalog number: P5619”.
Na2CO3 (EMD Millipore Corporation, catalog number: 106392 )
Copper(II) sulfate pentahydrate (CuSO4.5H2O) (Sigma-Aldrich, catalog number: 209198 )
Potassium sodium tartrate tetrahydrate (Sigma-Aldrich, catalog number: 217255 )
Folin-Ciocalteu’s phenol reagent (EMD Millipore Corporation, catalog number: 109001 )
Potassium phosphate monobasic (KH2PO4) (BD Biosciences, Falcon®, catalog number: 60229 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 60229”.
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, Falcon®, catalog number: 56814 )
KCl (EMD Millipore Corporation, catalog number: 104936 )
Ethylendiaminetetraacetic acid disodium salt-2-hydrate (EDTA) (Riedel-de Haën, catalog number: 34549 )
Note: Currently, it is “Sigma-Aldrich, FLUKA, catalog number: 34549 ”.
Sucrose (BD Biosciences, Falcon®, catalog number: 84100 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 84100”.
K2HPO4.3H2O (AppliChem GmbH, catalog number: 122333 )
Nevirapine (Viramune) (Boehringer Ingelheim)
Methanol, HiPerSolv CHROMANORM for HPLC-Gradient Grade (VWR International, Prolabo, catalog number: 20864.320 )
Dimethyl sulfoxide (DMSO) (minimum 99.5% GC, plant cell culture tested) (Sigma -Aldrich, catalog number: D4540 )
Nortriptyline hydrochloride, minimum 98% TLC (Sigma-Aldrich, catalog number: N7261 )
D-Glucose 6-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G7250 )
β-Nicotinamide adenine dinucleotide phosphate disodium salt (β-NADP) (Sigma-Aldrich, catalog number: 93205 )
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma-Aldrich, catalog number: G5760 )
MgCl2.6H2O, BioReagent, suitable for cell culture, suitable for insect cell culture (Sigma-Aldrich, catalog number: M2393 )
Acetonitrile (VWR, Prolabo, catalog number: 83639.320 )
Human liver microsomes pooled from 50 different individual donors (Thermo Fisher Scientific, InvitrogenTM, catalog number: HMMCPL )
Sodium hydroxide (extra pure, pellets) (Scharlab,S.L., catalog number: SO0420005P )
Ethyl acetate (extra pure) (Scharlab,S.L., catalog number: AC01431000 )
NaH2PO4.2H2O (Guinama, catalog number: 90790 )
Triethylamine (Sigma-Aldrich, catalog number: T0886 )
50 mM sodium-potassium phosphate buffer (see Recipes)
0.1 M potassium phosphate buffer (see Recipes)
Lowry reagent (see Recipes)
50 mM sodium phosphate monobasic buffer (see Recipes)
Equipment
Surgical tools
Scissors
Spectrophotometer (Hitachi High-Technologies Corporation, model: U-2900 )
1 cm polystyrene spectrophotometer cuvette (Sigma-Aldrich, catalog number: C5219 )
Thermostatic water bath
Refrigerated centrifuge (Sigma Laborzentrifugen, model: 2K15 )
Refrigerated ultracentrifuge (130,000 rpm) (Beckman Coulter, model: OptimaTM MAX ultracentrifuge )
Tissue Grinder, Potter-Elvehjem type, 30 ml Glass Vessel and Plain Plunger (VWR, Prolabo, catalog number: 432-0204 and 89026-400 , respectively)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Usach, I. and Peris, J. (2015). In vitro Studies: Inhibition of Nevirapine Metabolism by Nortriptyline in Hepatic Microsomes. Bio-protocol 5(19): e1607. DOI: 10.21769/BioProtoc.1607.
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Category
Microbiology > Antimicrobial assay > Antiviral assay
Microbiology > Microbial biochemistry > Other compound
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1,608 | https://bio-protocol.org/exchange/protocoldetail?id=1608&type=0 | # Bio-Protocol Content
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Peer-reviewed
Chick Neural Tube Explant Culture
Zhanna Alekseenko
Elisabet Andersson
José M. Dias
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1608 Views: 9986
Edited by: Oneil G. Bhalala
Reviewed by: Pamela MaherGeoff Lau
Original Research Article:
The authors used this protocol in Dec 2014
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Dec 2014
Abstract
The neural tube explant culture technique allows in vitro culturing of small pieces of neural tissue isolated from e.g., chick or mouse embryonic tissue in a matrix of collagen for defined periods of time. This method can be used to study the effects of defined molecules on developmental processes such as neural progenitor proliferation and neuronal differentiation and/or survival. Since the explant material can also be prepared from embryonic tissue electroporated with expression vectors, this technique can be adapted to study gene function in the presence of specific environmental signals. Different regions of the neural tube can also be isolated during the dissection step, allowing specific regions of the neural tube to be studied separately. Here, we present a neural tube explant culture method that we have used in several studies (Dias et al., 2014; Lek et al., 2010; Vallstedt et al., 2005).
Materials and Reagents
10 cm Petri dish (SARSTEDT AG & Co, catalog number: 82.1473 )
Glass Pasteur pipette 150 mm (VWR International, catalog number: 612-1701 )
Transfer pipette (SARSTEDT AG & Co, catalog number: 86.1174 )
Tungsten wire (Goodfellow, catalog number: w005160 )
10 ml syringe (HARTMANN USA, Omnifix®, catalog number: 4617100V )
Needle 20 G (Henke Sass Wolf, catalog number: 4710009040 )
Fresh fertilized chick eggs (ideally from a local supplier)
L-15 medium (Life Technologies, catalog number: 11415-049 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11415-049”.
Dispase I (Roche Diagnostics, catalog number: 04942086001 )
Heat inactivated Fetal bovine serum (FBS) ( Life Technologies, GibcoTM, catalog number: 10106-169 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 10106-169 ”.
Dulbecco’s modified eagle’s medium 10x (MEM) (Sigma-Aldrich, catalog number: D2429 )
Sodium hydroxide (NaOH) (EMD Millipore Corporation, catalog number: 1.06469.1000 )
Collagen (3.1 mg/ml) (Pure Col) (Advanced BioMatrix, catalog number: 5005-B )
Sodium bicarbonate 7.5% (BIC) (Life Technologies, catalog number: 25080 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25080”.
DMEM/F-12, GlutaMAXTM supplement (Life Technologies, catalog number: 31331-028 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 31331-028”.
Neurobasal medium (Life Technologies, catalog number: 21103-049 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21103-049”.
Albumin from bovine serum (BSA) (Sigma-Aldrich, catalog number: A7906 )
Glutamax (Life Technologies, catalog number: 35050-038 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 35050-038”.
Penicillin Streptomycin (PEST) (Life Technologies, catalog number: 15140-122 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
N2-supplement (Life Technologies, catalog number: 17502-048 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048”.
B27-supplement (Life Technologies, catalog number: 17504-044 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044”.
2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Dispase I solution (see Recipes)
Bedding solution (see Recipes)
Growing media (see Recipes)
Equipment
Humidified, 38 °C egg incubator (Grumbach, catalog number: 8012 )
Humidified, 37 °C tissue culture incubator (Thermo Fisher Scientific, catalog number: 51030287 )
16 °C incubator (Gemini BV Laboratory，Liebherr, catalog number: 4294 F1-1 )
Dissection microscope and light source (ZEISS, models: Zeiss Stemi SV11 and Zeiss KL1500LCD )
Borosilicate glass capillaries (Harvard Apparatus, catalog number: GC100TF-10 )
Nunc 4-well dishes for IVF (Thermo Fisher Scientific, catalog number: 144444 )
Flaming/brown micropipette puller (SUTTER, catalog number: P-97 )
Needle holder (Fine science tools, catalog number: 26016-12 )
Tweezers (Fine science tools, catalog number: 11252-20 )
Micro-scissors (Fine science tools, catalog number: 15000-00 )
Dissection scissors (Fine science tools, catalog number: 14088-10 )
Mouth aspirator system/Aspirator tube assemblies for calibrated microcapillary pipettes (Sigma-Aldrich, catalog number: A5177-5EA )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Alekseenko, Z., Andersson, E. and Dias, J. M. (2015). Chick Neural Tube Explant Culture. Bio-protocol 5(19): e1608. DOI: 10.21769/BioProtoc.1608.
Download Citation in RIS Format
Category
Neuroscience > Development > Explant culture
Cell Biology > Tissue analysis > Tissue isolation
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1,609 | https://bio-protocol.org/exchange/protocoldetail?id=1609&type=0 | # Bio-Protocol Content
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Peer-reviewed
Histochemical Staining of Silica Body in Rice Leaf Blades
RY Ryusuke Yokoyama
NK Natsumi Kido
TY Tsuyoshi Yamamoto
JF Jun Furukawa
HI Hiroaki Iwai
SS Shinobu Satoh
KN Kazuhiko Nishitani
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1609 Views: 10479
Edited by: Tie Liu
Reviewed by: Yuko Kurita
Original Research Article:
The authors used this protocol in Feb 2015
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The authors used this protocol in:
Feb 2015
Abstract
Silicon (Si) is a biologically important element for plants in the order Poales (Yamamoto et al., 2011; Kido et al., 2015). In rice, Si is mainly deposited in the motor cells and the cell walls of the leaf epidermis. However, the molecular basis of this overall process has not been elucidated. Thus, we propose a protocol for the histochemical staining of the silica body based on specific hydrogen bonding between silanol group and the carboxylate group of crystal violet lactone (Ichimura et al., 2008), as described by Isa et al. (2010), but with minor modifications. This modified protocol can be used for observing Si accumulation during rice development.
Keywords: Rice Cell wall Silica body Crystal violet lactone Motor cell
Materials and Reagents
Rice (Oryza sativa L. cv. Nipponbare) plants were grown in a liquid medium supplemented with 1.5 mM SiO32- in a growth chamber at 28 °C under a 15/9 h light/dark cycle (light at 150 μ mol-2 s-1). This protocol is performed with the fifth leaf blade at foliar age 5.2 (Kido et al., 2015). The foliar age “m.n” is defined as those in which the mth leaf is fully expanded and the (m+1)th leaf is under development with a length of n/10 of the fully expanded-length
Paraformaldehyde (Wako Pure Chemical Industries, catalog number: 162-16065 )
Sodium cacodylate buffer (Nacalai Tesque, catalog number: 37238-25 )
Agar powder (Nacalai Tesque, catalog number: 01028-85 )
Phosphate Buffered Saline (PBS) (Takara Bio, catalog number: T900 )
Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
Molecular sieves (Sigma-Aldrich, catalog number: M6141 )
Benzene (Nacalai Tesque, catalog number: 04017-35 )
Crystal violet lactone (Tokyo Chemical Industry UK Ltd, catalog number: C0741 )
PBS tablets (Takara Bio, catalog number: T900)
Phosphate-buffered saline (PBS) (see Recipes)
100% ethanol solution (see Recipes)
Crystal violet lactone solution (see Recipes)
Equipment
Growth chamber (Nippon Medical & Chemical Instruments, model: LH220S )
Stirrer Hotplate (Thermo Fisher Scientific, model: Fisher Scientific Isotemp )
Diaphragm vacuum pump (Leybold-Heraeus, model: Divac 2.2L )
Desiccator (SANPLATEC, model: PC-250K )
Microwave oven (Sharp Electronics, model: RE-T12 )
Paraffin dish (Greiner Bio-One GmbH, catalog number: 908177 )
Leica VT1200S vibrating blade microtome (Leica Microsystems, model: VT1200S )
Microscope slide (Matsunami Glass, catalog number: S-2123 )
Cover slip (Matsunami Glass, catalog number: C024361 )
Optical microscope (Leica Microsystems, model: DMRPX )
CCD camera (QImaging, model: Retiga EXi )
Procedure
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How to cite：Yokoyama, R., Kido, N., Yamamoto, T., Furukawa, J., Iwai, H., Satoh, S. and Nishitani, K. (2015). Histochemical Staining of Silica Body in Rice Leaf Blades. Bio-protocol 5(19): e1609. DOI: 10.21769/BioProtoc.1609.
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Category
Plant Science > Plant physiology > Ion analysis
Plant Science > Plant cell biology > Cell staining
Biochemistry > Other compound > Ion
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161 | https://bio-protocol.org/exchange/protocoldetail?id=161&type=0 | # Bio-Protocol Content
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Total RNA Extraction from Formalin-Fixed, Paraffin-Embedded (FFPE) Blocks
ZM Zhihai Ma
Published: Vol 2, Iss 7, Apr 5, 2012
DOI: 10.21769/BioProtoc.161 Views: 29333
Original Research Article:
The authors used this protocol in Sep 2009
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Abstract
Total RNA is extracted from fixed biological specimens by this method with higher yield than commercial kits. The product contains intact micro RNAs and small RNAs, and fragmented long RNAs.
Materials and Reagents
Paraffin embedded specimen
100% xylene (Thermo Fisher Scientific, catalog number: 6601 )
100% ethanol
Protease K (Life Technologies, Ambion®, catalog number: AM2546 )
Trizol (Life Technologies, Invitrogen™, catalog number: 15596-018 )
Chloroform (Sigma-Aldrich, catalog number: C2432 )
Glycogen (F. Hoffmann-La Roche, catalog number: 10901393001 )
Isopropyl alcohol (Isopropanol)
RNase-free water or TE buffer (USB, catalog number: 75834 ; Promega Corporation, catalog number: P1193 )
Tris-HCl (Life Technologies, Invitrogen™, catalog number: 15568-025 )
CaCl2 (Sigma-Aldrich, catalog number: C5670 )
Sodium dodecyl sulfate (Life Technologies, Invitrogen™, catalog number: 15525-017 )
Commercial kit (Life Technologies, Ambion®, Austin, TX)
Sodium dodecyl sulfate
Protease digestion buffer (see Recipes)
Equipment
Microtome
Microcentrifuge
Siliconized tubes (Thomas Scientific, catalog number: 2591L12 )
Procedure
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Category
Molecular Biology > RNA > RNA extraction
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1,610 | https://bio-protocol.org/exchange/protocoldetail?id=1610&type=0 | # Bio-Protocol Content
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Quantification of Callose Deposition in Plant Leaves
Loredana Scalschi
Eugenio Llorens
GC Gemma Camañes
VP Victoria Pastor
EF Emma Fernández-Crespo
VF Victor Flors
PG Pilar García-Agustín
BV Begonya Vicedo
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1610 Views: 15042
Edited by: Tie Liu
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
Callose is an amorphous homopolymer, composed of β-1, 3-glucan, which is widespread in higher plants. Callose is involved in multiple aspects of plant growth and development. It is synthetized in plants at the cell plate during cytokinesis, in several stages during pollen development and is deposited at plasmodesmata to regulate the cell-to-cell movement of molecules. Moreover, it is produced in response to multiple biotic and abiotic stresses (Chen and Kim, 2009). Callose is considered to act as a physical barrier by strengthening the plant cell well to slow pathogen infection and to contribute to the plant’s innate immunity. Thus the callose staining method is useful to quantify activity of plant immunity. In addition, this staining can be used to visualize structures in plant tissue, where the callose may be implied whether during the development of plants or response against pathogen infection. This method is based on the use of methyl blue which reacts with (1→3)-β-glucans to give a brilliant yellow fluorescence in UV light. Moreover, calcofluor stains chitin present in fungal cell membranes and also binds to cellulose at locations where the cuticle is damaged.
Keywords: Callose Papillae Plant resistance 1,3-beta-glucans Penetration resistance
Materials and Reagents
Plant materials: Arabidopsis thaliana cotyledons and leaves, Solanum lycopersicum leaves or Citrus leaves
96% ethanol
Na2HPO4.2H2O
NaH2PO4.2H2O
Methyl blue (Sigma-Aldrich, catalog number: M6900 )
Sodium phosphate buffer (see Recipes)
Methyl blue solution 0.5% and 0.05% (see Recipes)
Optional
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
Fluorescent Brightener 28 (synonym Calcofluor white) (Sigma-Aldrich, catalog number: F3543 )
HCl
Tris.HCl buffer (see Recipes)
Fluorescent brightener solution 0.01% (see Recipes)
Equipment
Leica IRB epifluorescence microscope with UV filter (BP 340 to 380 nm, LP 425 nm) equipped with a Leica DC300F camera (Leica Microsystems, model: Leica IRB )
Software
Digital photographs analysis software like GIMP (http://www.gimp.org) or Adobe Photoshop (adobe.com) are recommended
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Scalschi, L., Llorens, E., Camañes, G., Pastor, V., Fernández-Crespo, E., Flors, V., García-Agustín, P. and Vicedo, B. (2015). Quantification of Callose Deposition in Plant Leaves. Bio-protocol 5(19): e1610. DOI: 10.21769/BioProtoc.1610.
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Category
Plant Science > Plant biochemistry > Other compound
Plant Science > Plant cell biology > Cell staining
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1,611 | https://bio-protocol.org/exchange/protocoldetail?id=1611&type=0 | # Bio-Protocol Content
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Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi
Jorge Verdín*
Eddy Sánchez-León*
RF Rosa Fajardo-Somera
CM Carlos A. Leal Morales
SB Salomón Bartnicki-García
MR Meritxell Riquelme
*Contributed equally to this work
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1611 Views: 14986
Edited by: Fanglian He
Reviewed by: Aksiniya AsenovaSadri Znaidi
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
Density gradient centrifugation has been utilized to characterize the subcellular distribution of physiologically relevant enzymes in yeasts and filamentous fungi (Leal-Morales et al., 1988; Martínez et al., 1989; Kamada et al., 1991). This approach is now potentiated by protein tagging and live imaging techniques, which make possible to relate a single protein with, for example, a discrete population of intracellular vesicles and their in vivo dynamics (Verdín et al., 2009; Fajardo-Somera et al., 2013; Sánchez-León et al., 2015). Here, we describe the density gradient centrifugation and fractionation analysis of cell-free homogenates of a Neurospora crassa (N. crassa) strain that expresses CHS-6 chitin synthase fused to the green fluorescent protein (Riquelme et al., 2007).
Keywords: Neurospora crassa Chitosomes Vesicles Fractionation
Materials and Reagents
Tygon R-3603 tubing, I.D. 1/16 inch (Saint-Gobain, catalog number: AAC00002 )
Grade 1 Qualitative Filter Papers (GE Healthcare, catalog number: 1001-090 )
Neurospora crassa conidia
Sucrose (Sigma-Aldrich, catalog number: S0389 )
Sodium phosphate monobasic (NaH2PO4.H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S369-500 )
Sodium phosphate dibasic anhydrous (Na2HPO4) (Sigma-Aldrich, catalog number: S-7907 )
Protease Inhibitor Cocktail, Complete ULTRA tablets EDTA-free (Roche Diagnostics, catalog number: 04693132001 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 04693132001 ”.
Glass beads (500 μm) (Bio Spec Product, catalog number: 11079105 )
10% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
65% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
D-Biotin (Faga Labs, catalog number: CAS-58-85-5 )
Vogel’s complete medium (see Recipes)
50x salt solution (see Recipes)
Trace element solution (see Recipes)
100 mM phosphate buffer (pH 8.2) (see Recipes)
Laemmli buffer (see Recipes)
Equipment
Braun MSK 50 ml shaking bottles (LABEQUIP LTD, catalog number: 8541302 )
VWR® Standard Hot Plate Stirrers (VWR International, catalog number: 12365-382 )
Quick-seal centrifuge tube (Beckman Coulter, catalog number: 344326 )
Shaker incubator (Lab-LineX, model: Orbit Environ Shaker )
Vacuum filtration system
Braun MSK cell homogenizer (LABEQUIP LTD, catalog number: 953030 )
Bright field microscope (Olympus, model: Vanox-S )
Beckman L8-70M Ultracentrifuge (pre-cooled at 4 °C) (Beckman Coulter)
Type 70Ti rotor (pre-cooled at 4 °C) (Beckman Coulter)
GM-40 Linear Gradient maker (C. B. S. Scientific)
Peristaltic (Multi-staltic) pump (Buchler Lab, catalog number: N/A )
Tube sealer (Beckman Coulter, catalog number: 342420 )
Density gradient fractionator (ISCO, 185)
Note: This model is not available anymore. Brandel BR-186 Gradient Fractionator with Syringe Pump can be used instead.
CIGNETTM Fraction collector (ISCO)
Note: This item is also discontinued, but Bio-Rad 2110 Fraction Collector can be used instead.
6505 W UV-Vis Spectrophotometer (Bibby-scientific, Jenway)
Refractometer (ZEISS, catalog number: 12230 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Verdín, J., Sánchez-León, E., Fajardo-Somera, R., Morales, C. A. L., Bartnicki-García, S. and Riquelme, M. (2015). Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi. Bio-protocol 5(19): e1611. DOI: 10.21769/BioProtoc.1611.
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Category
Microbiology > Microbial biochemistry > Protein
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1,612 | https://bio-protocol.org/exchange/protocoldetail?id=1612&type=0 | # Bio-Protocol Content
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Sample Preparation for Correlative Light and Electron Microscopy (CLEM) Analyses in Cellular Microbiology
VL Viktoria Liss
MH Michael Hensel
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1612 Views: 16640
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Sep 2014
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Abstract
Dynamic processes in cells are usually monitored by live cell fluorescence microscopy. Unfortunately, this method lacks the ultrastructural information about the structure of interest (SOI). Currently, electron microscopy (EM) is the best tool to achieve highest spatial resolution. In addition, correlative light and electron microscopy (CLEM) analysis of the same structure allows combining authentic live cell imaging with the resolution power of EM. Additionally the reference space of the SOI is revealed. Our CLEM analyses of HeLa cells allow tracing the morphology and dynamic behavior of intracellular micro-compartments in living cells and their ultrastructure and subcellular organization in a highly resolved manner.
Keywords: Sample preparation Correlative Electron microscopy Eucaryotic cells Protocol
Materials and Reagents
General lab equipment: Gloves, lab coat, pipettes, 15 and 50 ml centrifuge tubes (e.g., BD Biosciences, Falcon®), microcentrifuge tubes (e.g., Eppendorf), plastic Pasteur pipettes, beakers
10 cm plastic Petri dish or a comparable vessel
3.5 cm plastic Petri dish or a comparable vessel (Acetone-resistant)
Eukaryotic cells as biological sample (here: transfected HeLa cells expressing LAMP1-GFP)
Cell-specific culture medium: DMEM (Biochrom AG, catalog number: FG 0445 ) + 10% Fetal Bovine Serum (FCS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270 )
Cell-specific imaging medium: MEM (Biochrom AG, catalog number: F 0475 ) + 30 mM HEPES
HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Carl Roth GmbH, catalog number: HN77.5 )
Glutaraldehyde [CH2(CH2CHO)2], 25% in H2O (Electron Microscopy Sciences, catalog number: E16221 )
Glycine, ultra pure (Biomol GmbH, catalog number: 04943.1 )
Large gelatin capsules, size 13 (Electron Microscopy Sciences, catalog number: 70114 )
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C 5670 )
Osmium tetroxide (OsO4), 0.1 g ampules (Electron Microscopy Sciences, catalog number: 19134 )
Ruthenium red [[(NH3)5RuORu(NH3)4ORu(NH3)5]Cl6] (AppliChem GmbH , catalog number: A34880001 )
Potassium hexacyanoferrate(III) [K3[Fe(CN)6]] (Sigma-Aldrich, catalog number: 244023 )
Ethanol p.a. grade (very pure chemical)
Acetone p.a. grade (very pure chemical)
EPON 812 (SERVA Electrophoresis GmbH, catalog number: 21045.02 )
DDSA = Dodecenylsuccinic anhydride (SERVA Electrophoresis GmbH, catalog number: 20755 )
MNA = Methylnadic anhydride (SERVA Electrophoresis GmbH, catalog number: 29452.03 )
DMP-30 = 2, 4, 6-Tris(dimethylamino-methyl)phenol (SERVA Electrophoresis GmbH, catalog number: 36975 )
Uranyl acetate dihydrate [UO2(CH3COO)2.2H2O] (Electron Microscopy Sciences, catalog number: 22400 )
Lead(II) nitrate [Pb(NO3)2] (Sigma-Aldrich, catalog number: 31137 )
Trisodium citrate dihydrate (Na3C6H5O7.2H2O) (Carl Roth, catalog number: 3580.1 )
1 N Sodium hydroxide (NaOH), carbonate-free (Electron Microscopy Sciences, catalog number: 21170-01 )
65% Nitric acid (HNO3)
Autoclaved ultrapure H2O (MilliQ)
Liquid nitrogen
Graded ethanol series (50%, 70%, 80%, 90%, 95%, 100%)
Mixes of acetone and EPON (3:1 and 1:3 mix)
1 M HEPES buffer (see Recipes)
2 M CaCl2 in H2O (see Recipes)
10% glycine in 0.2 M HEPES buffer (see Recipes)
1% ruthenium red in H2O (see Recipes)
15% potassium hexacyanoferrate(III) in H2O (see Recipes)
2x fixative (see Recipes)
Post-fixative (see Recipes)
EPON resin (see Recipes)
2% uranyl acetate in H2O (see Recipes)
Lead citrate according to Reynolds (see Recipes)
3% nitric acid (HNO3) (see Recipes)
Equipment
Cell culture equipment: Cell culture incubator (37 °C, 5% CO2, 90% humidity) and clean bench
MatTek Glass Bottom Culture Dishes, 35 mm, uncoated, Glass No. 2, gridded (Glass Bottom Dishes, MatTek Corporation, catalog number: P35G-2-14-CGRD )
Attention: Glass No. 2 may be too thick for some objectives with high numerical aperture (NA), but this is the only format available and worked in our application.
Confocal laser-scanning microscope (CLSM) Leica SP5 equipped with an incubation chamber (homemade) maintaining 37 °C and humidity during live cell imaging (Attention: CO2-containing atmosphere can be omitted if using imaging medium buffered with 30 mM HEPES. For carbonate-buffered cell culture media, the atmosphere should contain 5% CO2). The microscope is operated with software package LAS-AF for setting adjustment, image acquisition and image processing
Several objectives, such as 10x (HC PL FL 10x, NA 0.3, DIC, dry), 20x (HC PL APO CS 20x, NA 0.7, DIC, dry), 40x (HCX PL APO CS 40x, NA 1.25-0.75, DIC, oil immersion) and 100x objective (HCX PL APO CS 100x, NA 1.4-0.7, DIC, oil immersion)
The polychroic mirror TD 488/543/633 for the three channels GFP/RFP/DIC (Leica Microsystems)
Fume hood
Ice or cold metal block
Scalpel
Oven, heatable to 60 °C
Dewar vessel for liquid nitrogen
Stereo microscope
Dark permanent marker
Vice
Jigsaw
Universal specimen holder for EPON blocks (Leica Microsystems, catalog number: 16701761 )
Double edge carbon steel blades (Plano, catalog number: 121-9 )
Ultramicrotome (Leica EM UC6)
Diamant knife ultra 45 °C, 2 mm (DIATOME, catalog number: DU4520 )
Formvar-coated EM cooper slit grids 2 x 1 mm (homemade coating) (Electron Microscopy Sciences, catalog number: G2010-Cu )
Forceps for EM grids, type 7 stainless steel (Plano GmbH, catalog number: T5039 )
Storage box for grids (Electron Microscopy Sciences, catalog number: G71138 )
Large forceps for liquid nitrogen and small forceps for embedding procedure
Automated staining device (nanofilm surface analysis ultrastainer)
TEM (ZEISS, model: EFTEM 902 A ), operated at 80 kV and equipped with a 2K wide-angle slow-scan CCD camera (Teacher Retirement System of Texas) with software ImageSP (Teacher Retirement System of Texas, model: image SysProg)
Software
ImageJ, http://rsbweb.nih.gov/ij/, Photoshop 5.5 (Adobe), or higher or similar software for stitching and overlay of images
Imaris (Bitplane), ImageJ, ZEN (ZEISS), LASAF (Leica Microsystems), or similar software for processing fluorescence images
Procedure
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Copyright: © 2015 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:
Liss, V. and Hensel, M. (2015). Sample Preparation for Correlative Light and Electron Microscopy (CLEM) Analyses in Cellular Microbiology. Bio-protocol 5(19): e1612. DOI: 10.21769/BioProtoc.1612.
Krieger, V., Liebl, D., Zhang, Y., Rajashekar, R., Chlanda, P., Giesker, K., Chikkaballi, D. and Hensel, M. (2014). Reorganization of the endosomal system in Salmonella-infected cells: the ultrastructure of Salmonella-induced tubular compartments. PLoS Pathog 10(9): e1004374.
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Category
Cell Biology > Cell imaging > Electron microscopy
Cell Biology > Cell imaging > Live-cell imaging
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1,613 | https://bio-protocol.org/exchange/protocoldetail?id=1613&type=0 | # Bio-Protocol Content
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Hydroponic Culture of ‘Micro-Tom’ Tomato
Reiko Motohashi
HE Harumi Enoki
CF Chikako Fukazawa
YK Yoshikazu Kiriiwa
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1613 Views: 14214
Edited by: Fanglian He
Reviewed by: Cindy Ast
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
We use ‘Micro-Tom’ to study tomato fruit ripening and development mechanisms. ‘Micro-Tom’ is suitable for cultivation and experiments due to its small size of 10 to 20 cm in height and short life cycle of 3 months. There is also an abundance of publically available information on ‘Micro-Tom’ including EST, full-length cDNA clones and transcriptome data. ‘Micro-Tom’ plants are grown in hydroponic culture under fluorescents using Arabidopsis cultural shelves in greenhouses or plant rooms to get data with reproducibility for transcriptome and proteome analyses.
Keywords: Tomato Micro-Tom Hydroponic culture
Materials and Reagents
‘Micro-Tom’ (Prof. Ezura and Prof. Mizoguchi of Tsukuba University, Solanum lycopersicum cv. ‘Micro-Tom’, a model plant in the Solanaceae family). ‘Micro-Tom’ is a dwarf phenotype cultivar, originally first reported of in 1989, fixed by crossbreeding Florida Basket and Ohio 4013-3 (12th filial generation) (Martí et al., 2006) (Figure 1)
Figure 1. Solanum lycopersicum cv. ‘Micro-Tom’
Nutrient solution [Ootsuka house 5 (OAT house 5) OATagurio Co. Ltd. 2776 (http://www.oat-agrio.co.jp/en/product/index.html)
MS (Murashige and Skoog) mineral salts (Wako Pure Chemical Industries, Siyaku, catalog number: 392-00991 )
Gamborg’s B-5 vitamin (Sigma-Aldrich, catalog number: G1019 )
Sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 193-00025 )
Agar (Bacto agar) (BD, catalog number: 214010 )
Nutrient solution (see Recipes)
MS (Murashige and Skoog) medium (Li, 2011) (see Recipes)
Equipment
Rock wool (a kind of mineral wool) (A0 25/40, 25 x 25 x 40 mm) (Nitoubou Co. Ltd. catalog number: 762-090 )
Blower (Air pump, 100 V, working pressure 0.012 MPa) (YASUNAGA CORPORATION, model: LP-30A ) (Figure 2 A)
Three-way tube (metal 6 mm caliber) (Nissei Sangyo Corporation, catalog number: 25424 ) (Figure 2B and 2C)
6 mm silicon tube (bore diameter 4 mm) (Figure 2B)
Air stone (17 x 17 x 60 mm) (Figure 2D)
Figure 2. Parts of hydroponic culture system. A. Blower, B. 6 mm silicon tube, C. Three-way tube, D. Air stone (17 x 17 x 60 mm).
Shallow container (Size accordingly to match the scale of your project. In this case we are using a container 10 cm deep, a good example is Tupperware. We should use light-proof type of containers, because they protect to occur algae.)
Perforated polystyrene foam board (2.5 cm thickness, 3.5 cm hole diameter, distance between holes is approximately 12 cm) (Figure 3)
Figure 3. Perforated Polystyrene Foam Board (2.5 cm thickness, 3.5 cm hole diameter)
Plant boxes (DUCHEFA Biochemie, Steri Vent Low container, catalog number: S1682.0048 and Standard Closure lids, catalog number: S1681.0032 )
Sponge (household daily-use type)
Procedure
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Copyright: © 2015 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:
Motohashi, R., Enoki, H., Fukazawa, C. and Kiriiwa, Y. (2015). Hydroponic Culture of ‘Micro-Tom’ Tomato. Bio-protocol 5(19): e1613. DOI: 10.21769/BioProtoc.1613.
Suzuki, M., Takahashi, S., Kondo, T., Dohra, H., Ito, Y., Kiriiwa, Y., Hayashi, M., Kamiya, S., Kato, M., Fujiwara, M., Fukao, Y., Kobayashi, M., Nagata, N. and Motohashi, R. (2015). Plastid proteomic analysis in tomato fruit development. PLoS One 10(9): e0137266.
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Category
Plant Science > Plant physiology > Plant growth
Plant Science > Plant physiology > Tissue analysis
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1,614 | https://bio-protocol.org/exchange/protocoldetail?id=1614&type=0 | # Bio-Protocol Content
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Coupling of HIV-1 gp120-derived Core Protein to Paramagnetic Beads and Adsorption Assays
JI Jidnyasa Ingale
RW Richard T Wyatt
Published: Vol 5, Iss 20, Oct 5, 2015
DOI: 10.21769/BioProtoc.1614 Views: 6758
Reviewed by: Savita Nair
Original Research Article:
The authors used this protocol in Dec 2014
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Abstract
Analysis of the functional activity in polyclonal serum following immunization of a complex protein or glycoprotein immunogen is a very important but tedious process. Fine mapping of epitope-specific antibodies is difficult when they are elicited at relatively low levels. In our recent study focused on developing an HIV-1 vaccine, we immunized rabbits with hyperglycosylated stable core immunogens, which were designed using high-resolution structural information to elicit antibodies against the primary receptor-binding, CD4-binding site on HIV-1 gp120. Using a solid phase adsorption assay, we could map the serum antibodies to the conserved CD4-binding site, a known broadly neutralizing determinant on exterior envelope glycoprotein, gp120.
Materials and Reagents
15 ml polystyrene tube (BD, Falcon, catalog number: 352096 )
Dynabeads® MyOneTM Tosylactivated (Thermo Fisher Scientific，Dynal Biotech, catalog number: 65501 )
Magnetic separator (Thermo Fisher Scientific，Dynal MPC, catalog number: 12301D )
Bovine serum albumin (BSA) (Biopioneer, catalog number: C0100 )
Recombinant protein (core gp120) to couple to the beads [expressed and purified in-house. Please check Ingale et al. (2014)]
Sodium azide (Sigma-Aldrich, catalog number: 438456 )
DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 10313-021 )
Fetal Bovine Serum (VWR International, Seradigm, catalog number: 1300-500 )
Protease inhibitor (Roche Diagnostics, catalog number: 11255500 )
Glycine-HCl (Sigma-Aldrich, catalog number: G2879 )
Sodium borate (AMRESCO, catalog number: 0390 )
Ammonium sulfate, 99+%, ACS reagent (Thermo Fisher Scientific, ACROS ORGANICS, catalog number: 42340-0010 )
Coupling buffer (see Recipes)
Blocking buffer (see Recipes)
Wash buffer (see Recipes)
Coating buffer (see Recipes)
Equipment
Magnetic separator (Thermo Fisher Scientific，Dynal MPC, catalog number: 12301D )
Absorbance reader (Molecular Devices, model: Spectramax Plus )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ingale, J. and Wyatt, R. T. (2015). Coupling of HIV-1 gp120-derived Core Protein to Paramagnetic Beads and Adsorption Assays. Bio-protocol 5(20): e1614. DOI: 10.21769/BioProtoc.1614.
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Category
Immunology > Antibody analysis > Antibody detection
Microbiology > Microbial biochemistry > Protein
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1,615 | https://bio-protocol.org/exchange/protocoldetail?id=1615&type=0 | # Bio-Protocol Content
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Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores
JI Jidnyasa Ingale
RW Richard T Wyatt
Published: Vol 5, Iss 20, Oct 5, 2015
DOI: 10.21769/BioProtoc.1615 Views: 7854
Reviewed by: Savita Nair
Original Research Article:
The authors used this protocol in Dec 2014
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Abstract
Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.
Materials and Reagents
96-well black bottom plates (Greiner Bio-One GmbH, catalog number: 655209 )
Anti-human Fc capture biosensors (Pall Corporation, ForteBio®, catalog number: 18-5060 )
Phosphate buffered saline (Life Technologies, GibcoTM, catalog number: 100-10023 )
Monoclonal antibodies and hyperglycosylated core analytes (expressed in-house)
Equipment
Octet Red system (Pall Corporation, ForteBio®, part number: 30-5048 )
Software
Data Analysis 6.2 evaluation software (Forte Bio)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ingale, J. and Wyatt, R. T. (2015). Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores. Bio-protocol 5(20): e1615. DOI: 10.21769/BioProtoc.1615.
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Category
Immunology > Antibody analysis > Antibody-antigen interaction
Microbiology > Microbial biochemistry > Protein
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1,616 | https://bio-protocol.org/exchange/protocoldetail?id=1616&type=0 | # Bio-Protocol Content
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Bacterial Porphyrin Extraction and Quantification by LC/MS/MS Analysis
SM Stefano Mancini
JI James A. Imlay
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1616 Views: 8896
Edited by: Valentine V Trotter
Original Research Article:
The authors used this protocol in May 2015
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May 2015
Abstract
Heme is an iron-containing porphyrin which acts as a prosthetic group in several enzymes involved in disparate functions, such as respiration and H2O2-scavenging. Escherichia coli is able to produce heme endogenously since it contains all the enzymes involved in the nine-step biosynthesis pathway, which in absence of stress and in iron-replete media proceeds unabated. However, we recently showed that two steps are affected by H2O2 stress (Mancini and Imlay, 2015). To compensate, two enzymes, namely the ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF), are activated by the H2O2-responsive regulator OxyR. Genetic mutations that block either adaptation cause the intracellular accumulation of protoporphyrin IX and coproporphyrinogen III, the substrates of HemH and HemF, respectively. We here describe a method used to extract and quantify protoporphyrin IX and coproporphyrin III, the product of the spontaneous oxidation of coproporphyrinogen III.
Keywords: Heme Protoporphyrin Protoporphyrinogen Ferrochelatase
Materials and Reagents
Bacterial cell culture
Ethyl acetate (Sigma-Aldrich, catalog number: 34972-1 L-R )
Acetic acid (Sigma-Aldrich, catalog number: 45754-100 ML-F )
Hydrochloric acid (Sigma-Aldrich, catalog number: H1758-100 ML )
Protoporphyrin IX (Frontier Scientific, catalog number: P562-9 )
Coproporphyrin III dihydrochloride (Frontier Scientific, catalog number: C-654-3 )
Formic acid (Sigma-Aldrich, catalog number: 14265-1ML )
Acetonitrile (Sigma-Aldrich, catalog number: 34967-250 ML )
Equipment
For porphyrin extraction
Sonicator (Fisher Scientific, model: 550 sonic dismembrator )
For the LC/MS/MS analysis
5500 QTRAP LC/MS/MS system (AB Sciex)
1200 series HPLC system (Agilent Technologies)
Degasser Autosampler (Agilent Technologies)
Binary pump (Agilent Technologies)
Zorbax SB-Aq column (4.6 x 50 mm, 5 µm) (Agilent Technologies, catalog number: 846975 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mancini, S. and Imlay, J. A. (2015). Bacterial Porphyrin Extraction and Quantification by LC/MS/MS Analysis. Bio-protocol 5(19): e1616. DOI: 10.21769/BioProtoc.1616.
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Category
Microbiology > Microbial biochemistry > Other compound
Biochemistry > Other compound > Porphyrins
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1,617 | https://bio-protocol.org/exchange/protocoldetail?id=1617&type=0 | # Bio-Protocol Content
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Analysis of in vivo Cellulose Biosynthesis in Arabidopsis Cells by Spinning Disk Confocal Microscopy
TV Tamara Vellosillo*
TY Trevor Yeats*
NS Nadav Sorek
*Contributed equally to this work
Published: Vol 5, Iss 19, Oct 5, 2015
DOI: 10.21769/BioProtoc.1617 Views: 8631
Edited by: Fanglian He
Reviewed by: Renate Weizbauer
Original Research Article:
The authors used this protocol in Jun 2006
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The authors used this protocol in:
Jun 2006
Abstract
Cellulose is a main component of plant cell walls. Tools to analyze cellulose mainly rely on analytical chemistry, which yields information about cellulose amounts and structure, but cannot be applied to intact tissues. Moreover, these methods measure total cellulose and cannot be used to assay cellulose synthesis per se. Live cell imaging of the catalytic subunits of the cellulose synthesis complex (CSC) conjugated to fluorescent proteins is an important tool to understand the dynamics of the cellulose biosynthesis process (Paredez et al., 2006). This method can be used in various genetic backgrounds (Sorek et al., 2014) or with different chemical inhibitors (Brabham and Debolt, 2012). Here we describe in detail the procedure to visualize the movement of CSCs at the plasma membrane. As the movement of CSCs is likely caused by glucan synthesis and extrusion into the cell wall, live cell analysis of CSC velocity provides a method to directly measure cellulose synthesis in vivo.
Keywords: Cellulose Cell wall Cellulose synthesis dynamics Live cell imaging Arabidopsis
Materials and Reagents
Microscope slides (25 x 76 x 1.0 mm) and #1.5 cover glass (24 x 30 mm)
Arabidopsis seedlings expressing functional fluorescent protein fusions to CESAs, the catalytic subunits of the CSC, such as GFP:CESA3 (Desprez et al., 2007), YFP:CESA6 (Paredez et al., 2006) or tdTomato:CESA6 (Sánchez-Rodríguez et al., 2012) under the control of their native promoters
Household bleach (Clorox)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 71727 )
Murashige and Skoog (MS) basal salts (Caisson Laboratories, catalog number: MSP01 )
2-(N-morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: RES0113M-B103X )
Sucrose (Fisher Scientific, catalog number: BP220 )
Agar (Sigma-Aldrich, catalog number: RES10020-A102X )
Vacuum Grease (Beckman Coulter)
0.5x Murashige and Skoog (MS) media (see Recipes)
Equipment
Growth chamber to grow plant material (e.g., Percival Scientific, model: CU-36L5 )
Square plates 90 x 90 x 15 mm
Spinning disk confocal head (Yokogawa Electric Corporation) mounted on a motorized inverted microscope (e.g., Leica Microsystems, model: Leica DMI6000 or Zeiss, model: Zeiss Cell Observer SD ), equipped with 488 and/or 561 nm excitation lasers and a Photometrics QuantEM 512SC Camera
Software
Software operating the confocal microscope (e.g., Metamorph, Molecular Devices)
ImageJ (http://imagej.nih.gov/ij/)
MultipleKymograph and WalkingAverage plugins for ImageJ (J. Reitdorf and A. Seitz, http://www.embl.de/eamnet/html/body_kymograph.html)
Imaris (BitPlane)
Excel (Microsoft)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Vellosillo, T., Yeats, T. and Sorek, N. (2015). Analysis of in vivo Cellulose Biosynthesis in Arabidopsis Cells by Spinning Disk Confocal Microscopy. Bio-protocol 5(19): e1617. DOI: 10.21769/BioProtoc.1617.
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Category
Plant Science > Plant biochemistry > Carbohydrate
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Confocal microscopy
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1,618 | https://bio-protocol.org/exchange/protocoldetail?id=1618&type=0 | # Bio-Protocol Content
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In vivo Fluorescein Isothiocyanate-dextran (FD4) Permeability Assay
CW Carey L Watson
MM Maxime M Mahe
MH Michael A. Helmrath
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1618 Views: 23875
Reviewed by: Ningfei An
Original Research Article:
The authors used this protocol in Nov 2014
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Nov 2014
Abstract
Using pluripotent stem cells, it is now becoming possible to develop tissue models of organ systems within the body. These organs will allow for the study of organ function, physiology, embryology, and even pathologic processes. Recently, our group developed a model of human small intestine developed from human pluripotent stem cells which when transplanted in vivo, produce a mature, cystic intestinal structure that has digestive functions similar to that of native small intestine (Watson et al., 2014). Intestinal permeability is a primordial function of both the epithelium and associated tight junctions to control nutrient intake and prevent the passage of pathogens. One way to study gastrointestinal paracellular permeability is by determining the ability of fluorophores-conjugated macromolecules (i.e., fluorescein isothiocyanate-dextran (FITC-dextran; or FD4) to cross from the lumen and into circulation (Dong et al., 2014). We were able to test the intestinal permeability by injecting FITC-dextran directly into the lumen of the bioengineered intestine and determining the fluorescence within the blood of the murine host at various time points after injection.
Materials and Reagents
EDTA blood collection tubes (BD Biosciences, catalog number: 365873 )
1 ml syringes
30 gauge needles
Microhematocrit Capillary Tubes (Thermo Fisher Scientific, catalog number: 22-362-566 )
1 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5510 )
96-well microplates (Greiner Bio-One GmbH, catalog number: 655101 )
Immune deficient NOD-SCID IL-2Rγnull (NSG) mice, 8-16 weeks of age (bred in house), transplanted with a bioengineered intestine (maturation of transplant then allowed for 6-8 weeks)
Fluorescein isothiocyanate-conjugated dextran (FITC-dextran 3-5 Kda) (Sigma-Aldrich, catalog number: FD4 )
Sterile water
Phosphate-buffered saline (PBS)
Isoflurane for inhaled anesthesia (Henry Schein, catalog number: 050033 )
Buprenorphine (Midwest Veterinary Supply, catalog number: 790.06700.3 )
Isopropyl alcohol
Povidone-Iodine
Small surgical kit (scissors, ringed forceps, needle driver/holder)
3-0 Vicryl suture (Ethicon US, catalog number: VR416 )
GLUture tissue adhesive 1.5 ml 1/Tb (Skin closure glue) (Henry Schein, catalog number: 034418 )
Equipment
Spectrophotofluorometer (BioTek Instruments, model: Synergy H1 )
Dissecting Stereomicroscope (Leica Microsystems)
Microcentrifuge
Hair clipper/trimmer
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cell Biology > Tissue analysis > Tissue staining
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1,619 | https://bio-protocol.org/exchange/protocoldetail?id=1619&type=0 | # Bio-Protocol Content
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Intravenous Tomato Lectin Injection to Assess Functional Vasculature
CW Carey L Watson
MM Maxime M Mahe
MH Michael A. Helmrath
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1619 Views: 11968
Reviewed by: Ningfei An
Original Research Article:
The authors used this protocol in Nov 2014
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The authors used this protocol in:
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Abstract
Pluripotent stem cells have recently allowed for the development of tissue models for the various organ systems within the body. These models allow scientists to study organ function, physiology, embryology, and even pathologic processes. Studies on tissue can be done in vitro and/or transplanted into animal models for studies in vivo. Recently, our lab developed a model of human small intestine derived from human pluripotent stem cells which when transplanted in vivo, matured into an intestinal structure similar to that of adult intestine. The maturity of the transplanted human intestinal tissue was dependent upon the development of an adequate blood supply primarily from the murine host. In order to better study the developed vascular network within our transplanted intestinal tissue, we injected Fluorescein labeled Lycopersicon esculentum (tomato) lectin into the mouse tail vein (Watson et al., 2014). Using the property of this lectin to bind to the endothelium, we were able to visualize the vasculature within the transplant.
Materials and Reagents
1 ml syringes
30 gauge needles
Small surgical kit (scissors, ringed forceps, needle driver/holder)
Immune deficient NOD-SCID IL-2Rγnull (NSG) mice, 8-16 weeks of age (bred in house), transplanted with a bioengineered intestine (maturation of transplant then allowed for 6-8 weeks)
Fluorescein labeled Lycopersicon esculentum lectin (Tomato-Lectin) (Vector Laboratories, catalog number: FL-1171 )
Phosphate-buffered saline (PBS)
Isopropyl Alcohol
4% Paraformaldehyde in PBS (PFA)
Water-based optical clearing solution (Ke et al., 2014)
Equipment
Dissecting Microscope (Leica Microsystems)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Watson, C. L., Mahe, M. M. and Helmrath, M. A. (2015). Intravenous Tomato Lectin Injection to Assess Functional Vasculature. Bio-protocol 5(20): e1619. DOI: 10.21769/BioProtoc.1619.
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Category
Cell Biology > Tissue analysis > Tissue staining
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162 | https://bio-protocol.org/exchange/protocoldetail?id=162&type=0 | # Bio-Protocol Content
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Alcian Blue – Alizarin Red Staining of Mouse Skeleton
Peichuan Zhang
Published: Vol 2, Iss 8, Apr 20, 2012
DOI: 10.21769/BioProtoc.162 Views: 31599
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Abstract
Our lab has used the Alcian blue – Alizarin red staining method with certain modifications to characterize skeleton deformities in mice lacking Pek/Perk, encoding a translational control eIF2alpha kinase.
Materials and Reagents
Neural buffered formalin (Sigma-Aldrich, catalog number: HT5014 )
Alcian blue 8GX (Sigma-Aldrich, catalog number: A5268 )
Trypsin (Sigma-Aldrich, catalog number: T1426 )
Saturated sodium borate (Sigma-Aldrich, catalog number: S9640 )
Alizarin red (Sigma-Aldrich, catalog number: A3882 )
Thymol (EM Life Science, catalog number: TX0615-1 )
Ethanol
Glacial acetic acid
Potassium hydroxide
Glycerol
KOH
Trypsin solution (see Recipes)
Equipment
Conical tube
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhang, P. (2012). Alcian Blue – Alizarin Red Staining of Mouse Skeleton. Bio-protocol 2(8): e162. DOI: 10.21769/BioProtoc.162.
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Category
Developmental Biology > Morphogenesis
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1,620 | https://bio-protocol.org/exchange/protocoldetail?id=1620&type=0 | # Bio-Protocol Content
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Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues
ED Etienne Delannoy
AL Andéol Falcon de Longevialle
CF Catherine Colas des Francs-Small
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1620 Views: 8575
Edited by: Tie Liu
Reviewed by: Elias Bassil
Original Research Article:
The authors used this protocol in Aug 2014
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Aug 2014
Abstract
This qPCR-based assay provides an overview of the expression levels of all mitochondrial transcripts (mRNAs and rRNAs) as well as splicing efficiency in Arabidopsis. It was developed before RNAseq techniques were widely used (de Longevialle et al., 2007), but is nevertheless still useful as it is cheaper to run and the analysis is much easier and faster to perform if the aim is only to look at mitochondrial transcripts. For intron-containing mRNAs, the use of primer sets specifically amplifying spliced or unspliced forms allows the evaluation of the splicing efficiency.
Keywords: Mitochondrial transcriptome Splicing assay Quantitative RT-PCR Mitochondria Arabidopsis thaliana
Materials and Reagents
Eppendorf-type microtubes (0.5 ml and 1.5 ml) (any brand)
LightCycler® 480 Multiwell plate (white) (Roche Diagnostics, catalog number: 04729749001 )
LightCycler® 480 Sealing Foil for qPCR run (Roche Diagnostics, catalog number: 04729757001 )
Adhesive PCR film seals for storing plates containing primer mixes (Thermo Fisher Scientific, catalog number: AB0558 )
Combitips 0.1 ml (Eppendorf, catalog number: 0030 089.405 or 0030 089.618 )
Arabidopsis thaliana plants grown either in vitro or in soil
RNeasy Plant Mini Kit (QIAGEN, catalog number: 74904 )
Water, for molecular biology, DNAse, RNAse and Protease free (Thermo Fisher Scientific, ACROS OrganicsTM, catalog number: 327390010 )
Ambion Turbo DNA-freeTM Kit (Life Technologies, catalog number: AM1907 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1907”.
3M Na acetate (pH 5.2)
100 % and 70 % ethanol
Agarose LE, analytical grade (Promega Corporation, catalog number: V3125 )
Primers for PCR and qPCR (see Table 1)
Table 1. Primers used in this protocol
Please click here for Table 1.
Taq polymerase
Note: any Taq polymerase can be used for this step, we use a home-purified enzyme.
dNTP set (Life Technologies, catalog number: 10297-018 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297-018”.
Superscript III Reverse transcriptase (Life Technologies, catalog number: 18080-044 )
Notes: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-044”.
This enzyme comes with 5x transcription buffer and a solution of 0.1 M DTT.
Random Hexanucleotide Primers (Life Technologies, catalog number: 48190011 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 48190011”.
RNaseOUT Recombinant Ribonuclease inhibitor (Life Technologies, catalog number:
10777-019 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777-019”.
LightCycler® 480 SYBR I Master mix (Roche Diagnostics, catalog number: 04887352001 )
Note: This reagent is light sensitive.
Equipment
Nanodrop UV-Vis Spectrophotometer (Thermo Fisher Scientific)
‘Multipette plus’ (Eppendorf, catalog number: 4981 000.019 )
LightCycler® 480 System (Roche Diagnostics)
Software
LightCycler® 480 Software version 1.5 (Roche Diagnostics, catalog number: 04994884001)
Procedure
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Copyright: © 2015 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:
Delannoy, E., Falcon de Longevialle, A. and Francs-Small, C. C. D. (2015). Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues. Bio-protocol 5(20): e1620. DOI: 10.21769/BioProtoc.1620.
Colas des Francs-Small, C., Falcon de Longevialle, A., Li, Y., Lowe, E., Tanz, S. K., Smith, C., Bevan, M. W. and Small, I. (2014). The pentatricopeptide repeat proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 are involved in the splicing of the multipartite nad5 transcript encoding a subunit of mitochondrial complex I. Plant Physiol 165(4): 1409-1416.
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Category
Plant Science > Plant molecular biology > RNA
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1,621 | https://bio-protocol.org/exchange/protocoldetail?id=1621&type=0 | # Bio-Protocol Content
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Product Analysis of Starch Active Enzymes by TLC
Darrell Cockburn
NK Nicole Koropatkin
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1621 Views: 12917
Edited by: Valentine V Trotter
Reviewed by: Laura Molina-García
Original Research Article:
The authors used this protocol in Jan 2015
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Abstract
Thin layer chromatography (TLC) is a useful technique for detecting the presence of monosaccharides through to oligosaccharides, though it needs to be optimized for the specific sugars that are analyzed. Here we present a method for visualizing the reaction product(s) of starch active enzymes, which can contain α-1, 4 linked and α-1, 6 linked glucose. This was first published in Molecular Microbiology (Cockburn et al., 2015). The TLC protocol is an adapted version of that published by Robyt and Mukerjea (Robyt and Mukerjea, 1994). For a summary of the products generated by starch active enzymes see the review by Hii et al. (2012).
Keywords: Thin Layer Chromatography Starch Amylase Maltooligosaccharides
Materials and Reagents
TLC Silica gel 60 F254 20 x 20 cm plates (Note 1) (Merck Millipore Corporation, catalog number: 1.05715.0001 )
Filter paper circles (15 cm diameter) (VWR International, catalog number: 470204-480 )
MilliQ quality water (dH2O)
Acetonitrile (anhydrous) (Sigma-Aldrich, catalog number: 271004 )
Ethyl acetate (anhydrous) (Sigma-Aldrich, catalog number: 270989 )
2-propanol (Sigma-Aldrich, catalog number: I9516 )
N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma-Aldrich, catalog number: 222488 )
Methanol (anhydrous) (Sigma-Aldrich, catalog number: 322415 )
Sulfuric acid (Fluka, catalog number: 84716 )
Note: Currently, it is “ Sigma-Aldrich, catalog number: 84716”.
Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128 )
α-D-Glucose (anhydrous) (Sigma-Aldrich, catalog number: 158968 )
Maltose monohydrate (Sigma-Aldrich, catalog number: M9171 )
Maltotriose (Sigma-Aldrich, catalog number: M8378 )
Maltotetraose, DP4 (Sigma-Aldrich, catalog number: 47877 )
Maltopentaose (TCI America, catalog number: M1023 )
Maltohexaose (Sigma-Aldrich, catalog number: M9153 )
Maltoheptaose (Sigma-Aldrich, catalog number: 284017 )
Isomaltose (Sigma-Aldrich, catalog number: I7253 )
D-Panose (Sigma-Aldrich, catalog number: P2407 )
Glycogen from bovine liver (Sigma-Aldrich, catalog number: G0885 )
Amylopectin from potato starch (Sigma-Aldrich, catalog number: A8515 )
Amylose from potato (Sigma-Aldrich, catalog number: A0512 )
Dextran from Leuconostoc spp. (Sigma-Aldrich, catalog number: 31389 )
Pullulan from Aureobasidium pullulans (Sigma-Aldrich, catalog number: P4516 )
Your enzyme of interest and appropriate buffer
Mobile phase (see Recipes)
Staining solution (see Recipes)
Oligosaccharide standards (see Recipes)
Glycogen solution (see Recipes)
Amylopectin solution (see Recipes)
Amylose solution (see Recipes)
Dextran solution (see Recipes)
Equipment
Note: All equipment needed is shown in Figure 1.
TLC developing chamber (VWR International, catalog number: 21432-739 )
Chromatography sprayer (Note 2) (Sigma-Aldrich, catalog number: Z529729 )
Oven capable of reaching 120 °C (Boekel Scientific, catalog number: 107905 )
(Optional) MilwaukeeTM Dual Temperature Heat Gun (Thermo Fisher Scientific, Fisher Scientific, catalog number: 19-313-598 )
CorningTM ScholarTM PC-170 Hotplate (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-641-564 )
Figure 1.Equipment needed for this protocol
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Carbohydrate > Polysaccharide
Biochemistry > Protein > Activity
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1,622 | https://bio-protocol.org/exchange/protocoldetail?id=1622&type=0 | # Bio-Protocol Content
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Peer-reviewed
Rat Aortic Ring Model to Assay Angiogenesis ex vivo
Isabelle Ernens
BL Bénédicte Lenoir
Yvan Devaux
DW Daniel R. Wagner
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1622 Views: 10533
Edited by: Ivan Zanoni
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
Angiogenesis is a multifactorial event which requires the migration, proliferation, differentiation and structure rearrangement of endothelial cells. This angiogenic process has been commonly studied using in vitro assays such as Boyden chamber assay, wound healing assay and tube formation assay. These assays mainly use monolayers of endothelial cells which are modified by repeated passages and are fully proliferative, a situation far away from physiology. In addition, not only endothelial cells are involved in this process but surrounding cells (such as pericytes, smooth muscle cells, fibroblasts) and the supporting matrix are also major players.
The three-dimensional ex vivo aortic ring model recapitulates the complexities of angiogenesis and combines the advantages of in vitro and in vivo models. The aortic ring is cultivated in a chemically defined culture environment. Microvessels which grow in this system are lumenized vessels with surrounding supporting cells and are essentially indistinguishable from microvessels formed during angiogenesis in vivo. The efficacy of pro-or anti-angiogenic factors can be determined in the absence of serum molecules which may otherwise interfere with the substances being tested (Nicosia and Ottinetti, 1990). However, this system requires access to fresh rat tissue but several samples can be prepared from one aorta.
Keywords: Aortic ring Angiogenesis Endothelial cells Ex vivo model
Materials and Reagents
Aluminium foils
Cell culture plates 48 well (VWR International, catalog number: 734-2157 )
Petri dishes (VWR International, catalog number: 734-2374 )
LEW/Ss NHsd males rats 8-10 weeks old (Harlan Laboratories)
Endothelial Cell Growth Medium (Lonza, catalog number: CC-3162 ) + 1% penicillin/streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
Collagen R (2 mg/ml) (SERVA Electrophoresis GmbH, catalog number: 47254 )
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S-5761 )
10x MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 21430-020 )
NaOH (Merck Millipore Corporation, catalog number: 1.06498 )
Equipment
Dissecting microscope (Motic Stereomicroscope K-400L)
Laminar flow hood
Inverted light microscopes
50 ml Beaker
25 mm magnetic bar
Magnetic stirrer
Bleue extra gillette razor blades
Scissors (Fine Science Tools, catalog number: 14090-11 )
Vannas-Tübingen spring scissors (Fine Science Tools, catalog number: 15003-08 )
2 Microdissection forceps (VWR International, catalog number: 734-0448 )
Dissecting boards and pins
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ernens, I., Lenoir, B., Devaux, Y. and Wagner, D. R. (2015). Rat Aortic Ring Model to Assay Angiogenesis ex vivo. Bio-protocol 5(20): e1622. DOI: 10.21769/BioProtoc.1622.
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Category
Cancer Biology > Angiogenesis > Animal models
Immunology > Animal model > Rat
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1,623 | https://bio-protocol.org/exchange/protocoldetail?id=1623&type=0 | # Bio-Protocol Content
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Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts
Joseph C. Chen
NR Nadia R. Roan
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1623 Views: 17307
Edited by: Andrea Introini
Reviewed by: Benoit Chassaing
Original Research Article:
The authors used this protocol in Jun 2014
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Abstract
Purification and culture of endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) from endometrial biopsies allows for downstream cell-specific in vitro studies. The utility of this protocol is the ease with which cells are purified without contamination from unwanted cell types, and the ability to use patient-paired eEC and eSF in experiments. These methods have been previously published, but here the protocol has been updated for maximum efficiency.
Keywords: Epithelial cells Stromal fibroblasts Human endometrium Endometrial biopsy Cell culture
Materials and Reagents
FalconTM 15 ml Conical Centrifuge Tubes (Thermo Fisher Scientific, catalog number: 14-959-49D )
Cell Culture/Petri Dishes (100 x 20 mm) (Thermo Fisher Scientific, NuncTM, catalog number: 172958 )
FalconTM 50 ml Conical Centrifuge Tubes (Thermo Fisher Scientific, catalog number: 14-432-22 )
Costar® 24 Well Clear TC-Treated Multiple Well Plates, Bulk Pack, Sterile (Corning, catalog number: 3527 )
Sterile Filtration Units (0.1 µm pore) (Merck Millipore Corporation, catalog number: SCVPU02RE )
Procurement of endometrial biopsies
Endometrial biopsies from reproductive age women (ages 28–53) were obtained as previously cited (Chen et al., 2013; Chen et al., 2014). Briefly, women who are undergoing benign gynecologic procedures are consented to donate a sample of their endometrial biopsies via the Committee on Human Research (CHR) at UCSF (CHR Protocol, catalog number: 10-02786 ). Each organization should ensure that the appropriate IRB protocols are being utilized.
Digestion media (see Recipes)
Collagenase I [1 gm/(249 U/mg)] (Worthington Biochemical Corporation, catalog number: LS004196 )
Hyaluronidase from sheep testes (856 U/mg solid) (Sigma-Aldrich, catalog number: H 2251 )
Note: This product has been discontinued. A comparable substitute is Sigma-Aldrich H2126 at equal kU/ml. It is possible to use other comparable products/classes of hyaluronidase as long as the U/mg is comparable and the product has comparable bioactivity.
HBSS w/ Mg2+ and Ca2+, pH ranging 6.7-7.8 (UCSF Cell Culture Facility)
HBSS w/o Mg2+ and Ca2+, pH ranging 6.7-7.8 (UCSF Cell Culture Facility)
Dulbecco’s phosphate buffered saline (pH 7.2) (PBS) (UCSF Cell Culture Facility)
Penicillin Streptomycin (Pen/Strep, 1 nM 1x working solution) (UCSF Cell Culture Facility)
Transfer media (see Recipes)
Fetal bovine serum (FBS) (Charcoal/Dextran Stripped, sterile filtered, virus and mycoplasma tested) (Gemin Bio-Productsi, catalog number: 100-119 )
MCDB-105 medium (powder) with trace elements (Sigma-Aldrich, catalog number: M6395 ) (see Recipes)
1N NaOH, cell culture grade (Sigma-Aldrich, catalog number: S2770 )
ddH2O
Pen/Strep
Stromal cell medium (SCM) (see Recipes)
DMEM without phenol red (Life Technologies, Gibco®, catalog number: 21063-029 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21063-029”.
Sodium pyruvate solution (1 mM working solution) (Sigma-Aldrich, catalog number: S8636 )
MCDB-105
FBS
Antibiotic Antimycotic (AB/AM, 1 nM 1x working solution) (UCSF Cell Culture Facility)
Gentamycin (gent, 0.1 nM 1x working solution) (UCSF Cell Culture Facility)
Cell culture materials and reagents
BioCoatTM Matrigel® Matrix Thin Layer 24 Well Clear Flat Bottom TC-Treated Multiwell Plate (Thin Layer 100 µg/cm2) (Corning, catalog number: 354605 )
40 µm sterile cell strainer (fits onto a 50 ml Falcon tube) (BD Biosciences, Falcon®, catalog number: 352340 )
Note: Currently, it is “ Corning, catalog number: 352340 ”.
Defined Keratinocyte Serum Free Media (KSFM) (Life Technologies, Gibco®, catalog number: 10785-012 ) [comes as part of a kit including the growth supplement (Life Technologies, Gibco®，catalog number: 10784-015 )]
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21063-029 and 10784-015”.
Accutase cell detachment solution (Merck Millipore Corporation, catalog number: SCR005 )
Red blood cell lysis buffer (see recipes)
Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 254134 )
Potassium bicarbonate (Sigma-Aldrich, catalog number: 237205 )
EDTA (500 mM stock) (Teknova, catalog number: E0306 )
PBS
0.25% Trypsin (UCSF Cell Culture Facility)
Equipment
Rotator (Multi-Rotator) (any rotator will do) (Biosan, model: Multi-bio RS-24 )
FisherbrandTM Cell Strainers (Thermo Fisher Scientific, catalog number: 22363547 )
Centrifuge (any appropriate centrifuge will do) (Thermo Fisher Scientific, model: Sorvall Legend RT + centrifuge)
Incubator (Thermo Fisher Scientific, model: Forma 3110 CO2 Water Jacketed Tissue Culture Incubator)
Serological pipettes
Sterile Forceps, scalpels, and other surgical tools (Thermo Fisher Scientific)
Note: Stainless steel, fully autoclavable and suitable for gamma radiation sterilization. The exact catalog numbers for the set currently used are unavailable but these items are similar across vendors.
Brightfield microscope (inverted for cell culture)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, J. C. and Roan, N. (2015). Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts. Bio-protocol 5(20): e1623. DOI: 10.21769/BioProtoc.1623.
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Category
Immunology > Mucosal immunology > Genitourinary tract
Cell Biology > Cell isolation and culture > Cell isolation
Cell Biology > Tissue analysis > Tissue isolation
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1,624 | https://bio-protocol.org/exchange/protocoldetail?id=1624&type=0 | # Bio-Protocol Content
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Peer-reviewed
Clonal Culture of Mouse Liver Progenitor Cells
NT Naoki Tanimizu
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1624 Views: 11503
Original Research Article:
The authors used this protocol in Dec 2014
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Abstract
Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and cholangiocytes. For analyzing their differentiation potential, clonal culture has been used for LPCs isolated by a cell sorter. In addition, we can use the culture to assess functions of target genes on differentiation potential of LPCs. This protocol describes the process of cell isolation and colony assay to examine proliferative and differentiation potential of LPCs.
Keywords: Bipotential Hepatocyte Cholangiocyte Liver Flow cytometry
Materials and Reagents
Autoclaved 250 μm Nylon mesh (Nippon Rikagaku Kikai)
Butterfly needle (23 gauge) (Terumo, catalog number: SV-23CLK )
FalconTM Cell Strainers (Falcon, catalog number: 332350 )
Note: Currently, it is “Thermo Fisher Scientific, Falcon™, catalog number: 332350 ”.
35 mm tissue culture dish (Corning, catalog number: 430165 )
Collagenase (used at 1 mg/ ml for perfusion) (Wako Pure Chemical Industries, catalog number: 032-10534 )
PBS
Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H9269 )
Ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid (Sigma-Aldrich, catalog number: E0396 )
Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
Hyaluronidase (Sigma-Aldrich, catalog number: H3566 )
Laminin 111 (BD biosciences, catalog number: 354232 )
Note: Currently, it is “Corning, catalog number: 354232”.
Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10 ml (BD biosciences, catalog number: 354230 )
Note: Currently, it is “Corning, catalog number: 354230”.
Recombinant Mouse Oncostatin M (OSM) Protein (R&D systems, catalog number: 495-MO )
EGF Recombinant Human Protein (Life technologies, catalog number: PHG0311 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: PHG0311”.
Recombinant Human HGF (NS0-expressed) Protein (R&D systems, catalog number: 294-HGN )
Insulin from bovine pancreas (Sigma-Aldrich, catalog number: I5500 )
Insulin/Transferrin/Selenium (ITS) (Life Technologies, catalog number: 41400-045 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 41400-045”.
Dexamethasone (Dex) (Sigma-Aldrich, catalog number: D-4902 )
Anti-CD16/32 antibody (Biolegend, catalog number: 101-301 )
FITC anti-Dlk1 antibody (MBL International, catalog number: D187-4 )
FITC anti-mouse CD326 (Ep-CAM) Antibody (Biolegend, catalog number: 118207 )
APC-Cy7 anti-CD45 (Biolegend, catalog number: 103-115 )
APC-Cy7-TER119 (Biolegend, catalog number: 116-223 )
PE-Cy7-anti-CD31 (Biolegend, catalog number: 102417 )
Paraformaldehyde (PFA)
Hoechst 33258 (Dojindo Molecular Technologies, catalog number: 343-07961 )
Anti-mouse albumin (ALB) antibody (Bethyl laboratories, catalog number: A90-134P )
Anti-cytokeratin 19 (CK19) (Tanimizu et al., 2003)
AlexaFluor 488 conjugated donkey anti-rabbit IgG (Life technologies, catalog number: A-21206 ) and AlexaFluor 555 conjugated donkey anti-goat IgG (Life Technologies, catalog number: A-21432)
Note: Currently, it is “Thermo Fisher Scientific, Novex™, catalog number: A-21206 and A-21432 ”.
Prolong Gold (Life Technologies, catalog number: P36930 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: P36930”.
L-15 medium (Sigma-Aldrich, catalog number: L4386 )
DMEM/nutrient mixture Ham F-21 (DMEM/F12) medium
Decomplemented Hyclone fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH30910.03 )
Note: Currently, it is “GE Healthcare, catalog number: SH30910.03”.
Niflumic acid (Sigma-Aldrich, catalog number: N0630 )
Blockace (DS Pharma Biomedical Co, catalog number: UK-B40 )
Cloning ring (ASAHI GLASS CO, catalog number: 11-016-006 )
Trypsin/EDTA (Sigma-Aldrich, catalog number: T4049 )
RNAiMAX (Life Technologies, catalog number: 13778030 )
Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 13778030”.
RNeasy Mini Kit (QIAGEN, catalog number: 74106 )
ROCK inhibitor (Wako Pure Chemical Industries, catalog number: 257-00511 )
Pre-perfusion solution (see Recipes)
Perfusion solution (see Recipes)
L-15 medium (see Recipes)
Hyaluronidase (see Recipes)
Culture medium (see Recipes)
Equipment
Centrifuge (KUBOTA)
FACSAriaII (BD biosciences)
CO2 incubator (The incubator is used to keep culture at 37 °C and under 5% CO2) (SANYO)
Fluorescence microscope (OLYMPUS, model: IX71 ) and digital camera (OLYMPUS, model: DP72 )
Plate reader Mutiskan JX (Labsystems)
ROCK inhibitor (Wako Pure Chemical Industries, catalog number: 257-00511 )
Round-shaped stirring bar (ASONE Corporation, catalog number: 1-5409-01 )
Procedure
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Category
Stem Cell > Adult stem cell > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell differentiation
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1,625 | https://bio-protocol.org/exchange/protocoldetail?id=1625&type=0 | # Bio-Protocol Content
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Isolation and Characterization Procedure for Indole Alkaloids from the Marquesan Plant Rauvolfia Nukuhivensis
NM Nicolas J. Martin
MN Maël Nicolas
GL Gaël Lecellier
Phila Raharivelomanana
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1625 Views: 11214
Edited by: Arsalan Daudi
Reviewed by: Zhaohui LiuSamik Bhattacharya
Original Research Article:
The authors used this protocol in Jan 2015
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Abstract
A plethora of natural products, mostly secondary metabolites, are isolated and purified from many different organisms, like plants, fungi, algae, marine invertebrates, etc. The extraction procedure is specific to each organism, but some guidelines are usually followed for any purification procedure regarding targeted metabolites, such as alkaloids. Alkaloids are secondary metabolites that contain basic nitrogen in their structures and they are often associated with interesting biological properties especially in pharmacology field. This protocol describes the isolation procedure of indole alkaloids from Rauvolfia nukuhivensis directly from the ethanol extract of the plant material yielding different skeleton-type compounds including non-basic derivatives (ajmaline, sarpagine, macroline and β-carboline). The procedure details the guidelines and the steps to characterize new or known isolated compounds, beginning from the plant collection to the molecule level with the use of spectroscopic techniques (NMR, MS, UV). We detailed the extraction and fractionation procedures followed by the purification of compounds, as well as their physico-chemical characterizations. The procedure is illustrated by the example of the purification of a large array of indole alkaloids from the bark of Rauvolfia nukuhivensis.
Materials and Reagents
Rauvolfia nukuhivensis (Voucher JFB 2808): In our example, the bark of Rauvolfia nukuhivensis was collected at Maauu in “Terre Déserte” area on Nuku Hiva Island, Marquesas archipelago, French Polynesia, at an altitude of 477 m and identified by Dr Jean-François Butaud. A voucher specimen (JFB 2808) has been deposited at the Herbarium of French Polynesia. The sample was dried after collection at 30 °C using an air dryer device and stored in the room until grinding and extraction.
Ethanol (95%) (Thermo Fisher Scientific, Fisher chemical)
Methanol (HPLC grade from Fisher chemical)
Dichloromethane (HPLC grade from Fisher chemical)
Cyclohexane (HPLC grade from Fisher chemical)
Ethyl acetate (HPLC grade from Fisher chemical)
Equipment
SEB Prep'line grinder (SEB)
Semi-preparative HPLC column Luna 5 μm C18 (50 mm x 10 mm) (Phenomenex, model: RP-C18 )
Vacuum liquid chromatography (Buchner filter from Buchi, chromatography performed with vacuum pump) (BÜCHI Labortechnik AG)
High Pressure Liquid Chromatography (HPLC) Waters 600 system equipped with a Waters 717 Plus autosampler, a Waters 998 photodiode array detector (WATERS, model: Waters 998 photodiode array detector ), and a Sedex 75 evaporative light-scattering detector (SEDERE, model: Sedex 75)
NMR spectrometer (Bruker Corporation, model: Bruker Avance 500 MHz )
Polarimeter (jascoinc, model: Jasco P-2000 )
Spectropolarimeter (jascoinc, model: Jasco J-810 )
Mass spectrometer (Bruker Corporation, model: Bruker ESQUIRE 3000 plus )
Thermo Finnigan LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Martin, N. J., Nicolas, M., Lecellier, G. and Raharivelomanana, P. (2015). Isolation and Characterization Procedure for Indole Alkaloids from the Marquesan Plant Rauvolfia Nukuhivensis. Bio-protocol 5(20): e1625. DOI: 10.21769/BioProtoc.1625.
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Category
Plant Science > Plant biochemistry > Other compound
Plant Science > Plant metabolism > Metabolite profiling
Biochemistry > Other compound > Alkaloid
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1,626 | https://bio-protocol.org/exchange/protocoldetail?id=1626&type=0 | # Bio-Protocol Content
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Collagen-induced Arthritis: A Model for Murine Autoimmune Arthritis
Kathryn M. Pietrosimone
Mengyao Jin
BP Brad Poston
Peng Liu
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1626 Views: 15033
Edited by: Ivan Zanoni
Original Research Article:
The authors used this protocol in Mar 2015
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Abstract
Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund’s adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund’s adjuvant (IFA) 21 days after the first injection. These mice typically develop disease 26 to 35 days after the initial injection. C57BL/6J mice are resistant to arthritis induced by type II bovine collagen, but can develop arthritis when immunized with type II chicken collagen in CFA, and receive a boost of type II chicken collagen in IFA 21 days after the first injection. The concentration of heat-killed Mycobacterium tuberculosis H37RA (MT) in CFA also differs for each strain. DBA/1J mice develop arthritis with 1 mg/ml MT, while C57BL/6J mice require and 3-4 mg/ml MT in order to develop arthritis. CIA develops slowly in C57BL/6J mice and cases of arthritis are mild when compared to DBA/1J mice. This protocol describes immunization of DBA/1J mice with type II bovine collagen and the immunization of C57BL/6J mice with type II chicken collagen.
Materials and Reagents
Interchangeable Syringes (Micro-Mate, catalog number: 148251A )
Note: Currently, it is “Thermo Fisher Scientific, Micro-Mate™, catalog number: 148251A ”.
Nylon 3-Way Stopcock (Kimble Chase, catalog number: 4201634503 )
Note: Currently, it is “Thermo Fisher Scientific, Kimble Chase™, catalog number: 4201634503 ”.
Needle (BD, catalog number: 305109 )
Disposable syringe (BD, catalog number: 309623 )
DBA/1J mice (male, 8 weeks old)
C57BL/6 (male, 8 weeks old)
Bovine Collagen Type II (Chondrex, catalog number: 20021 )
Chicken Collagen Type II (Chondrex, catalog number: 20011 )
Complete Freund's Adjuvant (CFA) (Chondrex, catalog number: 7008 -1 mg/ml and 7001 -4 mg/ml)
Incomplete Freund's Adjuvant (IFA) (Sigma-Aldrich, catalog number: F5506 )
Ketamine (KetaVed) (Vedco, catalog number: 078908598 )
Xylazine (AnaSed) (LLOYD, catalog number: 078081939 )
Glacial acetic acid (Thermo Fisher Scientific, catalog number: A38-212 )
Equipment
Filtration System (Corning, catalog number: 431096 )
Tailveiner Restrainer (Braintree Scientific, catalog number: TV-150 )
Caliper (Kafer, catalog number: 217901 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pietrosimone, K. M., Jin, M., Poston, B. and Liu, P. (2015). Collagen-induced Arthritis: A Model for Murine Autoimmune Arthritis. Bio-protocol 5(20): e1626. DOI: 10.21769/BioProtoc.1626.
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Category
Immunology > Animal model > Mouse
Immunology > Inflammatory disorder > Arthritis
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1,627 | https://bio-protocol.org/exchange/protocoldetail?id=1627&type=0 | # Bio-Protocol Content
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Hyaloperonospora arabidopsidis (Downy Mildew) Infection Assay in Arabidopsis
Shuta Asai
Ken Shirasu
Jonathan D. G. Jones
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1627 Views: 16240
Edited by: Arsalan Daudi
Original Research Article:
The authors used this protocol in Oct 2014
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Abstract
Hyaloperonospora arabidopsidis (Hpa; formerly Peronospora parasitica or Hyaloperonospora parasitica) is an oomycete downy mildew pathogen of the model plant Arabidopsis. The pathosystem between Arabidopsis and Hpa has been extensively used to study host/pathogen co-evolution (Coates and Beynon, 2010). As Hpa is an obligate biotrophic pathogen, its host is absolutely required for survival. Thus, Hpa must be maintained on susceptible Arabidopsis accessions and mutants. Growth of Hpa is evaluated in two ways; counting conidiospores (Asai et al., 2014) or counting sporangiophores after trypan blue staining (Holt et al., 2005). Here, we describe how to do inoculation with Hpa and how to evaluate Hpa growth on Arabidopsis.
Keywords: Downy mildew Arabidopsis Oomycete Hyaloperonospora arabidopsidis
Materials and Reagents
Materials and Reagents1.Conical tubes (50 ml) (BD Biosciences, Falcon®, catalog number: 352070 )
Miracloth (Calbiochem®, catalog number: 475855 )
Note: Currently, it is “EMD Millipore Corporation, catalog number: 475855”.
Labeling Tape (Shamrock, catalog number: ST20 )
Paper towels
Arabidopsis lines and Hpa isolate. Ws-2 eds1-1 mutants [the accession previously reported as Ws-0 is in fact Ws-2 (Parker et al., 1996)] and Col-0 plants were used as susceptible and resistant accessions of Arabidopsis for Hpa isolate Emoy2, respectively.
Ethanol (70%)
Plastic tray (270 x 270 x 60 mm) with a transparent lid
Sterile water
Bright-LineTM Hemacytometer (Sigma-Aldrich, catalog number: Z359629 )
Trypan blue (Sigma-Aldrich, catalog number: T6146 )
Phenol (Wako Pure Chemical Industries, Siyaku, catalog number: 160-12725 )
Chloral hydrate (Sigma-Aldrich, catalog number: C8383 )
Glycerol (Wako Pure Chemical Industries, Siyaku, catalog number: 07500616 )
Trypan blue solution (see Recipes)
Chloral hydrate solution (see Recipes)
Equipment
Scissors
Biological safety cabinet (Labconco, model: Purifier Delta Series Class II Type A2 Cabinet )
Airbrush Kit (Airtex, model: ASCF4 and KIDS105 )
Growth chamber (NKsystem) (Nippon Medical & Chemical Instruments Co., catalog number: LPH410S )
Weight (Sartorius, model: Quintix 224-1S )
Water bath ( NE1-8 ) (Thermo Fisher Scientific, catalog number: 11459499)
Labo shaker (BIO CRAFT, catalog number: BC730 )
Stereomicroscope (Leica Microsystems, catalog number: M165FC )
Procedure
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Copyright: © 2015 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:
Asai, S., Shirasu, K. and Jones, J. D. G. (2015). Hyaloperonospora arabidopsidis (Downy Mildew) Infection Assay in Arabidopsis. Bio-protocol 5(20): e1627. DOI: 10.21769/BioProtoc.1627.
Asai, S., Rallapalli, G., Piquerez, S. J., Caillaud, M. C., Furzer, O. J., Ishaque, N., Wirthmueller, L., Fabro, G., Shirasu, K. and Jones, J. D. (2014). Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid. PLoS Pathog 10(10): e1004443.
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Category
Plant Science > Plant immunity > Disease bioassay
Plant Science > Plant immunity > Perception and signaling
Microbiology > Microbe-host interactions > In vivo model
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1,628 | https://bio-protocol.org/exchange/protocoldetail?id=1628&type=0 | # Bio-Protocol Content
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Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells
SR Stefanie Rosa
PS Peter Shaw
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1628 Views: 7704
Edited by: Tie Liu
Reviewed by: Vinay Panwar
Original Research Article:
The authors used this protocol in Dec 2014
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Abstract
Chromatin-binding proteins play a crucial role in chromatin structure and gene expression. Direct binding of chromatin proteins both maintains and regulates transcriptional states. It is therefore important to study the binding properties of these proteins in vivo within the natural environment of the nucleus. Photobleaching, photoactivation and photoconversion (photoswitching) can provide a non-invasive experimental approach to study dynamic properties of living cells and organisms. We used photoactivation to determine exchange dynamics of histone H2B in plant stem cells of the root (Rosa et al., 2014). The stem cells of the root are located in the middle of the tissue, which made it impossible to carry out photoactivation of sufficiently small and well-defined sub-cellular regions with conventional laser illumination in the confocal microscope, mainly because scattering and refraction effects within the root tissue dispersed the focal spot and caused photoactivation of too large a region. We therefore used 2-photon activation, which has much better inherent resolution of the illuminated region. This is because the activation depends on simultaneous absorption of two or more photons, which in turns depends on the square (or higher power) of the intensity-a much sharper peak. In this protocol we will describe the experimental procedure to perform two-photon photoactivation experiments and the corresponding image analysis. This protocol can be used for nuclear proteins tagged with photoactivable GFP (PA-GFP) expressed in root tissues.
Keywords: Histone exchange Two-Photon Microscopy FRAP Plant stem cells
Materials and Reagents
Sterile 9 cm square Petri dishes for plant growth media
Cover slips No 1.5 (VWR International, catalog number: 631-0125 )
Microscope slides with frosted end (VWR International, Superfrost®, catalog number: 631-0114 )
Secure Seal Adhesive Sheets (0.12 mm thick) (Grace Bio-Labs, Inc., catalog number: 620001 )
Arabidopsis lines expressing a protein of interest tagged with PA-GFP (Patterson and Lippincott-Schwartz, 2002)
Note: In our case, for measuring histone exchange dynamics in the root stem cells we placed the H2B-PAGFP construct under the control of a root cell-specific promoter (pSCR) (Rosa et al., 2014), which derives expression simultaneously in quiescent centre cells, endodermis/cortex initials, and root endodermis. This setup allowed us to compare the dynamics of a histone protein in pluripotent stem cells as they progress into more differentiated states. In this protocol we will not mention particular cells or tissues as the procedure can be applied to any cell type in the root.
Murashige & Skoog Basal Medium with Vitamins (any equivalent source would be suitable) (PhytoTechnology Laboratories®, catalog number: M519 )
10% bleach (contains 5-10% sodium hypochlorite) in dH2O (Vortex, Procter & Gamble)
NH4NO3 (pH 5.8) (ForMediumTM)
Sucrose (Sigma-Aldrich)
Phytagel (Sigma-Aldrich, catalog number: P8169 )
Murashige and Skoog medium (see Recipes)
Equipment
Plant growth chamber
Two-photon photoactivation experiments were performed on an Ultima two-photon laser scanning microscope (Buker Coopration, Prairie Technologies, model: Ultima 2-Photon Microscope )
Note: Live images were acquired using Olympus 60x 0.9 NA water immersion objectives at 512 x 512 resolution (0.203 μm/pixel) and 1 μm focal steps.
Laminar flow cabinet
Software
ImageJ software
MS Excel
Graphpad prism software
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Rosa, S. and Shaw, P. (2015). Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells. Bio-protocol 5(20): e1628. DOI: 10.21769/BioProtoc.1628.
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Category
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Two-photon microscopy
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1,629 | https://bio-protocol.org/exchange/protocoldetail?id=1629&type=0 | # Bio-Protocol Content
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Vacuole Structure Analysis during Cell Death Subsequent to Application of Erwinia carotovora Culture Filtrates to Cell Cultures of Nicotiana tabacum
Yumi Hirakawa
Seiichiro Hasezawa
Takumi Higaki
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1629 Views: 7499
Edited by: Samik Bhattacharya
Reviewed by: Daniel Savatin
Original Research Article:
The authors used this protocol in Jan 2015
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Abstract
We recently established an experimental model system for efficient defense-related cell death using tobacco BY-2 cultured cells treated with culture filtrates of the pathogenic bacterium Erwinia carotovora (E. carotovora) (Hirakawa et al., 2015). Applying this experimental system to transgenic BY-2 cells stably expressing the vacuolar membrane marker GFP-VAM3 (Kutsuna and Hasezawa, 2002) allowed us to monitor changes in vacuolar membrane structures including a decrease of transvacuolar strands during cell death (Hirakawa et al., 2015). Our model system can help to investigate organelle dynamics in defense-related cell death. Here, we show protocol for applying E. carotovora filtrates to BY-2 cells and confocal observation of vacuolar membrane dynamics and subsequent cell death. We used cell cycle synchronized BY-2 cells to effectively monitor invaginated vacuolar membranes such as transvacuolar strands in our recent report (Hirakawa et al., 2015); however, we do not describe the protocol for cell cycle synchronization in this article. For the step-by-step protocol for BY-2 cell synchronization, please refer to previous protocol papers (Nagata and Kumagai, 1999; Kumagai-Sano et al., 2006).
Keywords: Cell death Vacuole Erwinia Tobacco Confocal microscopy
Materials and Reagents
Sterile filter with pore size 0.22 µm (Merck Millipore Corporation, Millex®–GV Filter unit, catalog number: SLGV033RS )
50 ml syringe (TERUMO CORPORATION, catalog number: SS-50ESZ )
12 well plate (Sumitomo Bakelite Co., catalog number: MS-80120 )
Erwinia carotovora subsp. carotovora (National Institute of Technology and Evaluation, catalog number: 103133)
Transgenic tobacco BY-2 cell culture (N. tabacum L. cv. Bright Yellow 2) stably expressing GFP-VAM3 (RIKEN Bioresource Center, catalog number: RPC00039 )
Aphidicolin (Wako Pure Chemical Industries, Siyaku, catalog number: 015-09814 )
Yeast extract (KANTO KAGAKU, KANTO Chemical, catalog number: 712021-5 )
BactoTM Tryptone (BD bioscience, catalog number: 211705 )
NaCl (KANTO KAGAKU, KANTO Chemical, catalog number: 37144-01 )
Murashige and Skoog plant salt mixture (Wako Pure Chemical Industries, Siyaku, catalog number: 392-00591 )
Sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 196-00015 )
myo-Inositol solution (Wako Pure Chemical Industries, Siyaku, catalog number: 094-00281 )
Thiamine hydrochloride (Wako Pure Chemical Industries, Siyaku, catalog number: 201-00852 )
Sodium 2,4-Dichlorophenoxyacetate Monohydrate (Tokyo Chemical Industry, catalog number: D1319 )
Lysogeny broth (LB) medium (see Recipes)
Modified Linsmaier and Skoog (LS) medium (see Recipes)
Equipment
Rotary shaker for E. carotovora culture (TAITEC CORPORATION, model: BR-40LF )
300 ml flask for E. carotovora culture (Sansyo, Iwaki, catalog number: 82-0087 )
Spectrophotometer (Beckman Coulter, model: DU® 640 )
Centrifuge (TOMY SEIKO CO, model: MX-300 )
Deep freezer (Nihon Freezer, catalog number: CLN-30U )
100 ml flask for BY-2 culture (Sansyo, Iwaki, catalog number: 82-0085 )
Rotary shaker for the untreated BY-2 culture (TAITEC CORPORATION, catalog number: 300LF )
Rotary shaker for the treated BY-2 culture (TAITEC CORPORATION, catalog number: NR-30 )
Incubator for the treated BY-2 culture (Sanyo, catalog number: MIR-553 )
Glass base dish (Sansyo, Iwaki, catalog number: 3911-035 )
Confocal laser scanning microscope (OLYMPUS, model: FV300 )
Procedure
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Category
Plant Science > Plant immunity > Disease bioassay
Microbiology > Microbe-host interactions > Bacterium
Microbiology > Microbe-host interactions > Ex vivo model
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163 | https://bio-protocol.org/exchange/protocoldetail?id=163&type=1 | # Bio-Protocol Content
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Immunofluorescence on Frozen Tissue Sections
JR Jason Reuter
Published: Dec 20, 2011
DOI: 10.21769/BioProtoc.163 Views: 30187
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Abstract
Immunofluorescence is commonly used to determine the cellular or tissue localization of a protein of interest. Immunofluorescence can also be used as a qualitative measure of protein expression.
Materials and Reagents
Primary antibody
Alexa fluor secondary antibodies (Life Technologies, InvitrogenTM)
Horse serum (Santa Cruz Biotechnology, catalog number: sc-2483 )
Hoechst 33342 nuclear dye (Life Technologies, InvitrogenTM, catalog number: H1399 )
ProLong gold antifade mounting reagent (Life Technologies, InvitrogenTM, catalog number: P36934 )
PAP pen (Sigma-Aldrich, catalog number: Z377821 )
Nail polish (Thermo Fisher Scientific, catalog number: 50-949-071 )
100% ice cold acetone
Phosphate buffered saline (PBS)
Formaldehyde
Methanol
Triton X-100
Equipment
-20 °C freezer
Humidified chamber
Procedure
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Reuter, J. (2011). Immunofluorescence on Frozen Tissue Sections. Bio-101: e163. DOI: 10.21769/BioProtoc.163.
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Category
Immunology > Immune cell staining > Immunodetection
Biochemistry > Protein > Fluorescence
Biochemistry > Protein > Expression
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1,630 | https://bio-protocol.org/exchange/protocoldetail?id=1630&type=0 | # Bio-Protocol Content
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Isolation of Triterpenes from Propolis (Bee Glue)
MK Muhammad N. Kardar
VS Veronique Seidel
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1630 Views: 13080
Edited by: Arsalan Daudi
Reviewed by: Cindy Ast
Original Research Article:
The authors used this protocol in Oct 2014
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The authors used this protocol in:
Oct 2014
Abstract
Propolis (bee glue) is a natural substance produced by bees upon collection of mainly plant resins. Bees use it as antiseptic sealing agent between honeycombs and to preserve the hive from external contamination. Numerous scientific studies have been published on the biological properties of propolis including its anti-inflammatory, anti-oxidant, immunostimulant, antitumour and antimicrobial activity. Different propolis chemotypes have been characterised based on the nature of the plant-derived substances present and the geographical origin of collection. Here, we describe the isolation of nine triterpenes from a sample of propolis originating from North-Western Cameroon. All compounds were identified following analysis of their spectroscopic data and comparison with previously published reports.
Materials and Reagents
TLC silica gel 60 F254 plates (VWR International, catalog number: 1.05554.0001 )
Wilmad® NMR tubes 5 mm diam., precision (Sigma-Aldrich, catalog number: Z274275 )
Snap-cap vials (VWR International, catalog number: 548-0555 )
Raw propolis (Nature’s Laboratory Ltd, catalog number: P5 )
Ethanol 96% v/v (extra pure, Specified Laboratory Reagent) (Thermo Fisher Scientific, catalog number: 10162252 )
Hexanes (for HPLC, 95% n-hexane approx) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10703611 )
Ethyl acetate, extra pure, Specified Laboratory Reagent (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10080130 )
Sodium sulfate anhydrous, extra pure (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10606082 )
Silica gel 60 (0.063–0.200 mm) (VWR International, catalog number: 1.07734.1000 )
Silica gel 60H (VWR International, catalog number: 1.07736.1000 )
98% p-Anisaldehyde (Sigma-Aldrich, catalog number: A88107 )
Acide sulfurique fumant (pure) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10222282 )
Acetic acid glacial (pure) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10375020 )
Methanol (extra pure, Specified Laboratory Reagent) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10214490 )
Sephadex® LH-20 (Sigma-Aldrich, catalog number: LH20100 )
α-Amyrin (Sigma-Aldrich, catalog number: 53017 )
Cycloartenol (Sigma-Aldrich, catalog number: 08172 )
Lupeol (Sigma-Aldrich, catalog number: S957712 )
Dichloromethane, extra pure, stabilised with amylene, Specified Laboratory Reagent (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10458210 )
Chloroform-d (CDCl3) (Sigma-Aldrich, catalog number: 10080130 )
Anisaldehyde-sulphuric acid reagent (see Recipes)
Equipment
Heated ultrasonic water bath (340 W) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10215332 )
Büchi rotary evaporator, Rotavapor® R-210 (VWR International, catalog number: 531-0850 )
UV viewing cabinet CC10 (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11778201 )
UV lamp UVG-11 230 V 50/60 Hz 4 W hand held short wave (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11718241 )
UV lamp UVL-21 230 V 50/60 Hz 4 W hand held long wave (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11728241 )
Analytical balance (VWR International, catalog number: 611-2267 )
Precision balance (VWR International, catalog number: 611-2695 )
Vacuum water jet pump (VWR International, catalog number: 181-9100 )
Pear-shaped (separating) funnel (1,000 ml) (Scientific Glass Laboratories, catalog number: SFP1L )
Round bottom flasks, 250 ml (Total number of 12) (Scientific Glass Laboratories, catalog number: FRS250/B19 or FRS250/B24 )
Glass foot measuring cylinder with hexagonal base, borosilicate glass, class “B” (25 ml and 250 ml) (Scientific Glass Laboratories, catalog number: MCB/25 and MCB/250 )
Conical Erlenmeyer flasks (500 ml) (Scientific Glass Laboratories, catalog number: FC500/B24 )
Filter funnels, borosilicate (Scientific Glass Laboratories, catalog number: BFF75 and BFF100 )
Rubber vacuum tubing NW8 (VWR international, catalog number: 189.3111 )
Glass Pasteur pipettes 150 mm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10006021 )
Glass Pasteur pipettes 230 mm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10209381 )
Glass micropipettes (homemade from glass Pasteur pipettes 230 mm using a Bunsen burner)
Büchner filter with cone and thread, GL14, for vacuum liquid chromatography (VLC) (Scientific Glass Laboratories, catalog number: TBF/4 / B24 / POR3 )
Twin trough chambers for TLC plates (VWR International, catalog number: 552-0011 )
Glass atomiser reagent sprayer (VWR International, catalog number: 552-0031 )
Glass chromatography columns (CC) with sintered discs (Scientific Glass Laboratories, catalog number: R2/40 )
Duratool DO1600 hot air heat gun (Amazon)
Whatman qualitative filter paper, grade 1 (Sigma-Aldrich, catalog number: WHA1001125 )
Exactive Orbitrap mass spectrometer (MS) operating in a positive and negative electrospray ionisation (ESI) switching mode (Thermo Fisher Scientific)
JEOL 505HA high resolution electron impact (HREI) mass spectrometer (MS) using direct probe at elevated temperature (110~160 ºC) at 70 eV (JEOL USA)
JEOL Lambda Delta 400 NMR spectrometer (JEOL USA, model: JEOL Nuclear Magnetic Resonance Spectrometers )
Software
Xcalibur software (version 2.2) for MS data processing (Thermo Fisher Scientific, model: Xcalibur software)
Mestre Nova (MNova) software (version 8.0.0) for NMR processing (Mestrelab Research SL, model: version 8.0.0)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kardar, M. N. and Seidel, V. (2015). Isolation of Triterpenes from Propolis (Bee Glue). Bio-protocol 5(20): e1630. DOI: 10.21769/BioProtoc.1630.
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Category
Plant Science > Plant biochemistry > Other compound
Biochemistry > Other compound > Terpene
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1,631 | https://bio-protocol.org/exchange/protocoldetail?id=1631&type=0 | # Bio-Protocol Content
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Peer-reviewed
Immune Cell Isolation from Mouse Femur Bone Marrow
Xiaoyu Liu
NQ Ning Quan
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1631 Views: 23769
Edited by: Oneil G. Bhalala
Reviewed by: Thomas X. Lu
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
The bone marrow is the site of hematopoiesis and contains mixed population of blood cells including erythrocytes, granulocytes, monocytes, dendritic cells, lymphocytes and hematopoietic stem cells. The following protocol provides a simple and fast method for isolation of bone marrow immune cells (no erythrocytes) from mouse femurs with a yield of approximate 8 x 107 cells in 5 ml culture media (1.6 x 104 cells/µl). Further isolation or flow cytometric analysis might be required for study of specific immune cell types.
Keywords: Blood Flow cytometry Hematopoietic stem cells
Materials and Reagents
Sterile paper towel
Sterile surgical pad (Direct Resource, catalog number: 19015742 )
23-gauge (or 25-/26-gauge) needle (BD Biosciences, catalog number: 305145 )
10 ml syringe (BD Biosciences, catalog number: 309604 )
70 µm nylon cell strainer (Falcon, catalog number: 352350 )
Note: Currently, it is “Corning, Falcon®, catalog number: 11995-065 ”.
50 ml conical tube (Falcon, catalog number: 21008-940 )
Note: Currently, it is “Corning, Falcon®, catalog number: 21008-940 ”.
5 ml syringe plunger (BD Biosciences, catalog number: 309646 )
Adult mice (> 6 weeks, any strain) (e.g., C57BL/6)
Hank’s balanced salt solution (HBSS), no Calcium, no Magnesium, no phenol red (Life Technologies, catalog number: 14175095 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14175095 ”.
DMEM medium, high glucose, pyruvate, L-glutamine (Life Technologies, catalog number: 11995-065 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065 ”.
70% ethanol
Fetal bovine serum heat inactivated (FBS) (Sigma-Aldrich, catalog number: F9665 )
Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 213330 )
Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 237205 )
Disodium edetate (Sigma-Aldrich, catalog number: D2900000 )
RBC lysis buffer (see Recipes)
DMEM medium (see Recipes)
Equipment
Blunt-end sterile scissors (Thermo Fisher Scientific, Fisher Scientific, catalog number: 08-950 )
Sharp sterile scissors (Thermo Fisher Scientific, Fisher Scientific, catalog number: 08-940 )
Sterile forceps (Thermo Fisher Scientific, Fisher Scientific, catalog number: 08-890 )
HausserTM LevyTM Hemacytometer Chamber Set (Thermo Fisher Scientific, Fisher Scientific, catalog number: 02-671-55A ) or coulter Z2 cell and particle counter (Beckman Coulter, catalog number: 383550 )
Refrigerated centrifuge
Sterile culture hood
CO2 rodent euthanasia chamber
Procedure
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Copyright: © 2015 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, X. and Quan, N. (2015). Immune Cell Isolation from Mouse Femur Bone Marrow. Bio-protocol 5(20): e1631. DOI: 10.21769/BioProtoc.1631.
Liu, X., Yamashita, T., Chen, Q., Belevych, N., McKim, D. B., Tarr, A. J., Coppola, V., Nath, N., Nemeth, D. P., Syed, Z. W., Sheridan, J. F., Godbout, J. P., Zuo, J. and Quan, N. (2015). Interleukin 1 type 1 receptor restore: a genetic mouse model for studying interleukin 1 receptor-mediated effects in specific cell types. J Neurosci 35(7): 2860-2870.
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Category
Immunology > Immune cell isolation
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1,632 | https://bio-protocol.org/exchange/protocoldetail?id=1632&type=0 | # Bio-Protocol Content
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Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
YM Yuta Mutaguchi
TO Toshihisa Ohshima
Published: Vol 5, Iss 20, Oct 20, 2015
DOI: 10.21769/BioProtoc.1632 Views: 7327
Reviewed by: Timo Lehti
Original Research Article:
The authors used this protocol in Nov 2013
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Nov 2013
Abstract
Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods for purification of this protein from L. otakiensis JCM 15040. Because no specific ligand that has high affinity for this enzyme has been identified, the purification was performed using ammonium sulfate fraction, four types of column chromatography and preparative Native-PAGE, not using an affinity column chromatography. We hope that the protocol will provide useful information for purification of an enzyme that cannot easily be purified using an affinity column chromatography.
Keywords: Lactic acid bacteria Protein purification Ion exchange chromatography Hydrophobic interaction chromatography Affinity chromatography
Materials and Reagents
1.5 ml tube (ASONE Corporation, catalog number: 2-1998-02 )
Dialysis membrane tube (molecular weight cutoff: 14,000) (EIDIA, catalog number: UC27-32-100 )
Amicon Ultra 15 ml centrifugal filter 3 K device (Merck Millipore Corporation, catalog number: UFC500396 )
Disposable homogenizer (1.5 ml scale) “Biomasher II” (NIPPI Corporation, catalog number: 320102 )
Feeding tube (TERUMO CORPORATION, catalog number: SF-ET1725 )
Syringe (10 ml scale) (TERUMO CORPORATION, catalog number: SS-10SZ )
Plug silicon (ASONE Corporation, catalog number: 6-336-03 )
Needle (TERUMO CORPORATION, catalog number: NN-2238R )
Purification of the isoleucine 2-epimerase
L. otakiensis JCM 15040 obtained from Japan Collection of Microorganisms (JCM)
TOYOPEARL Phenyl-650M column
Note: Pack 50 ml of TOYOPEARL Phenyl-650M resin in a chromatography column (diameter: 2.5 cm, length: 10 cm) (Tosoh Bioscience LLC, catalog number: 14478 ).
TOYOPEARL Butyl-650M column
Note: Pack 50 ml of TOYOPEARL Butyl-650M resin in a chromatography column (diameter: 2.5 cm, length: 10 cm) (Tosoh Bioscience LLC, catalog number: 07477 ).
TOYOPEARL SuperQ-650M column
Note: Pack 50 ml of TOYOPEARL SuperQ-650M resin in a chromatography column (diameter: 2.5 cm, length: 10 cm) (Tosoh Corporation, catalog number: 17227 ).
Acrylamide (Wako Pure Chemical Industries, Siyaku, catalog number: 016-00765 )
N, N’-Methylenebisacrylamide (Nacalai Tesque, catalog number: 22402-02 )
Ammonium persulfate (Wako Pure Chemical Industries, Siyaku, catalog number: 7727-54-0 )
N, N, N’, N’-Tetramethylethylenediamine (Nacalai Tesque, catalog number: 33401-72 )
Ammonium sulfate (Wako Pure Chemical Industries, Siyaku, catalog number: 019-03435 )
Sodium chloride (Wako Pure Chemical Industries, Siyaku, catalog number: 198-01675 )
Lactobacilli MRS Broth (MRS medium powder) (BD, catalog number: 288130 ) (see Recipes)
50 mM sodium phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM dithiothreitol (see Recipes)
Note: Unless otherwise indicated, this buffer is used as the standard buffer throughout the purification procedures.
Sodium dihydrogenphosphate dihydrate (Wako Pure Chemical Industries,
Siyaku, catalog number: 192-02815 )
Disodium hydrogenphosphate 12-water (Wako Pure Chemical Industries,
Siyaku, catalog number: 196-02835 )
Ethylenediamine-N, N, N', N'-tetraacetic acid, disodium salt, dehydrate (EDTA)
(Dojindo Molecular Technologies, catalog number: N001 )
Dithiothreitol (Nacalai Tesque, catalog number: 14128-04 )
Red Sepharose CL-4B column (Ohshima and Sakuraba, 1986) (see Recipes)
Note: According to Recipes, prepare Red Sepharose CL-4B resin by attaching Reactive Red 120 (Sigma-Aldrich, catalog number: R0378-50G ) to Sepharose® CL-4B (Sigma-Aldrich, catalog number: CL4B200-100ML ). Pack 10 ml of Red Sepharose CL-4B resin in a chromatography column (diameter: 1.5 cm, length: 12 cm).
1.5 M Tris-HCl buffer (pH 8.8) (see Recipes)
2-Amino-2-hydroxymethyl-1, 3-propanediol (Tris base) (Wako Pure Chemical Industries, Siyaku, catalog number: 207-06275 )
Hydrochloric acid (Nacalai Tesque, catalog number: 18402-45 )
0.5 M Tris-HCl buffer (pH 6.8) (see Recipes)
2-Amino-2-hydroxymethyl-1, 3-propanediol (Tris base)
Hydrochloric acid
Native-PAGE electrophoresis buffer (see Recipes)
2-Amino-2-hydroxymethyl-1, 3-propanediol (Tris base)
Glycine (Wako Pure Chemical Industries, Siyaku, catalog number: 077-00735 )
2x Native-PAGE gel loading buffer (see Recipes)
2-Mercaptoethanol (Nacalai Tesque, catalog number: 21418-42 )
Sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 193-00025 )
Bromophenol blue (KANTO CHEMICAL, catalog number: 04319-30 )
Isoleucine 2-epimerase activity assay
Pyridoxal-5’-phosphate (Nacalai Tesque, catalog number: 29606-74 )
L-isoleucine (PEPTIDE INSTITUTE, catalog number: 2712 )
Flavin adenine dinucleotide disodium salt (Nacalai Tesque, catalog number: 16010-06 )
4-Aminoantipyrine (Nacalai Tesque, catalog number: 01907-52 )
Phenol (Wako Pure Chemical Industries, Siyaku, catalog number: 168-12721 )
D-Amino acid oxidase from porcine kidney (Sigma-Aldrich, catalog number: A5222-200UN )
Peroxidase (TOYOBO BIO CHEMICAL DEPT, catalog number: PE0-301 )
0.5 M sodium phosphate buffer (pH 8.0) (see Recipes)
Sodium dihydrogenphosphate dihydrate
Disodium hydrogenphosphate 12-water
Equipment
Purification of the isoleucine 2-epimerase
Incubator (TITEC CORPORATION, model: BR-43FM )
Centrifuge (Hitachi Koki Co., model number: CR 21F and TOMY SEIKO CO., model number: MX-300 )
Multi-Beads shocker (Yasui Kikai Corporation)
Electrophoresis apparatus
Column chromatography equipment (see Representative data)
Magnetic stirrer (ASONE Corporation, model: HS-50E )
Two-way stopcock (Bio-Rad Laboratories, catalog number: 7328102 )
Three-way stopcock (TERUMO CORPORATION, catalog number: TS-TR1K )
Chromatography column (diameter: 2.5 cm, length: 10 cm) (Bio-Rad Laboratories, catalog number: 7372512 )
Chromatography column (diameter: 1.5 cm, length: 12 cm) (Bio-Rad Laboratories, catalog number: 7321010 )
Fraction tube (15 ml scale) (ASONE Corporation, catalog number: 2-8007-02 )
Isoleucine 2-epimerase activity assay
Spectrophotometer (Shimadzu Scientific Instruments, model number: UVmini-1240 )
Water bath (TOKYO RIKAKIKAI CO., model number: NTT-20S )
Vortex (TAITEC CORPORATION, catalog number: 0061271-000 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mutaguchi, Y. and Ohshima, T. (2015). Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040. Bio-protocol 5(20): e1632. DOI: 10.21769/BioProtoc.1632.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Isolation and purification
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1,633 | https://bio-protocol.org/exchange/protocoldetail?id=1633&type=0 | # Bio-Protocol Content
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Determination of Fructokinase Activity from Zobellia galactanivorans
Agnès Groisillier
Thierry Tonon
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1633 Views: 9336
Edited by: Valentine V Trotter
Reviewed by: Christian Roth
Original Research Article:
The authors used this protocol in Mar 2015
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The authors used this protocol in:
Mar 2015
Abstract
Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g.,, the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant fructokinase (FK) of Zobellia galactanivorans. The ZgFK enzyme catalyzes the conversion of fructose to fructose-6-phosphate using ATP as a cofactor.
[Principle] Fructokinase (FK) activity was determined by an enzyme-coupled assay (Figure 1). ADP formed through the FK reaction, i.e., phosphorylation of fructose to fructose-6-phosphate (F6P), is used by the pyruvate kinase (PK) which transforms phosphoenolpyruvate (PEP) to pyruvate. Then, lactate dehydrogenase (LDH) converts pyruvate to lactate using NADH as a cofactor. FK activity is measured by following the decrease in absorbance at 340 nm which corresponds to the transformation of NADH to NAD+.
Figure 1. Enzyme-coupled reaction used for determination of fructokinase (FK) activity
Keywords: Mannitol degradation Fructokinase Zobellia galactanivorans Flavobacteria
Materials and Reagents
UV-Star® PS Microplate (96 Well) (Greiner Bio-One GmbH, catalog number: 655801 )
Purified recombinant His-tagged ZgFK
Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ZgFK vector, as described by Groisillier et al. (2010). This recombinant protein was purified by affinity chromatography using a HisPrep FF 16/10 column (GE Healthcare Dharmacon) onto an Äkta avant system (GE Healthcare Dharmacon). The complete purification protocol is described in details in Groisillier et al. (2015).
Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle (Sigma-Aldrich, catalog number: P0294 )
MilliQ water
Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt (Sigma-Aldrich, catalog number: P7252 )
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
4-morpholineethane-sulfonic acid (MES) (Sigma-Aldrich, catalog number: M2933 )
Bis-Tris (Sigma-Aldrich, catalog number: B9754 )
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
Examples of chemicals to be tested to assess substrate specificity:
D-(-)-fructose (Sigma-Aldrich, catalog number: F0127 )
D- (+)-glucose (Sigma-Aldrich, catalog number: G8270 )
D-(+)-mannose (Sigma-Aldrich, catalog number: M4625 )
D-mannitol (Sigma-Aldrich, catalog number: M9647 )
D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
D-mannitol-1-phosphate (Sigma-Aldrich, catalog number: 92416 )
D-fructose-1-phosphate (Sigma-Aldrich, catalog number: S408689 )
α-D-Glucose 1-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G9380 )
D-Mannose 6-phosphate sodium salt (Sigma-Aldrich, catalog number: M3655 )
D-Glucose 6-phosphate sodium salt (Sigma-Aldrich, catalog number: G7879 )
D-Fructose 6-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: F3627 )
Adenosine 5’-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, catalog number: A1852 )
1 M Tris-HCl (pH 7.5) (see Recipes)
100 mM MgCl2 (see Recipes)
1 M KCl (see Recipes)
100 mM PEP (see Recipes)
100 mM ATP (see Recipes)
10 mM NADH (see Recipes)
PK/LDH (see Recipes)
Equipment
Safire2 UV spectrophotometer microplate reader (Tecan Trading AG)
NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific)
Software
Hyper 32 (Hyper32.software.informer.com)
Microsoft Excel
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Groisillier, A. and Tonon, T. (2015). Determination of Fructokinase Activity from Zobellia galactanivorans. Bio-protocol 5(21): e1633. DOI: 10.21769/BioProtoc.1633.
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Category
Microbiology > Microbial biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Mannitol
Biochemistry > Protein > Activity
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1,634 | https://bio-protocol.org/exchange/protocoldetail?id=1634&type=0 | # Bio-Protocol Content
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Peer-reviewed
Determination of Mannitol-2-dehydrogenase Activity
Agnès Groisillier
Thierry Tonon
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1634 Views: 8564
Edited by: Valentine V Trotter
Reviewed by: Christian Roth
Original Research Article:
The authors used this protocol in Mar 2015
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Mar 2015
Abstract
Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g., the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) of Zobellia galactanivorans. The ZgM2DH enzyme catalyzes the reversible conversion of mannitol to fructose using NAD+ as a cofactor. ZgM2DH activity was assayed in both directions, i.e., fructose reduction and mannitol oxidation.
Reversible reaction:
D-mannitol + NAD+ ↔ D-fructose + NADH + H+
Keywords: Mannitol degradation Mannitol-2-dehydrogenase Flavobacteria Zobellia galactanivorans
Materials and Reagents
UV-Star® PS Microplate (96 Well) (Greiner Bio-One GmbH, catalog number: 655801 )
Purified recombinant His-tagged ZgM2DH
Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ZgM2DH vector, as described by Groisillier et al. (2010). This recombinant protein was purified by affinity chromatography using a HisPrep FF 16/10 column (GE Healthcare Dharmacon) onto an Äkta avant system (GE Healthcare Dharmacon). The complete purification protocol is described in details in Groisillier et al. (2015).
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
MilliQ water
4-morpholineethane-sulfonic acid (MES) (Sigma-Aldrich, catalog number: M2933 )
Bis-Tris (Sigma-Aldrich, catalog number: B9754 )
Examples of chemicals to be tested to assess substrate and co-factor specificity:
D-(−)-fructose (Sigma-Aldrich, catalog number: F0127 )
D-(+)-glucose (Sigma-Aldrich, catalog number: G8270 )
D-(+)-mannose (Sigma-Aldrich, catalog number: M4625 )
D-(+)-galactose (Sigma-Aldrich, catalog number: G0750 )
D-(+)-xylose (Sigma-Aldrich, catalog number: X1500 )
D-mannitol (Sigma-Aldrich, catalog number: M9647 )
D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
D-(+)-arabitol (Sigma-Aldrich, catalog number: A3381 )
β-Nicotinamide adenine dinucleotide hydrate (β-NAD) (Sigma-Aldrich, catalog number: N1636 )
β-Nicotinamide adenine dinucleotide phosphate hydrate (β-NADP) (Sigma-Aldrich, catalog number: N5755 )
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (β-NADH) (Sigma-Aldrich, catalog number: N8129 )
β-Nicotinamide adenine dinucleotide phosphate, reduced tetra (cyclohexylammonium) salt (β-NADPH) (Sigma-Aldrich, catalog number: N5130 )
1 M Tris-HCl (pH 7.5) (see Recipes)
10 mM NADH (see Recipes)
10 mM NAD+ (see Recipes)
Equipment
Safire2 UV spectrophotometer microplate reader (Tecan Trading AG)
NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific)
Software
Hyper 32 (hyper32.software.informer.com)
Microsoft Excel
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Groisillier, A. and Tonon, T. (2015). Determination of Mannitol-2-dehydrogenase Activity . Bio-protocol 5(21): e1634. DOI: 10.21769/BioProtoc.1634.
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Category
Microbiology > Microbial biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Mannitol
Biochemistry > Protein > Activity
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1,635 | https://bio-protocol.org/exchange/protocoldetail?id=1635&type=0 | # Bio-Protocol Content
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Isolation and Flow-cytometric Analysis of Mouse Intestinal Crypt Cells
DA Dayanira Alsina-Beauchamp
PR Paloma del Reino
AC Ana Cuenda
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1635 Views: 15751
Edited by: HongLok Lung
Reviewed by: Kristina Y. Aguilera
Original Research Article:
The authors used this protocol in Nov 2014
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Abstract
The intestinal epithelial layer forms tubular invaginations into the underlying connective tissue of the lamina propria. These structures, termed crypts, are the basic functional unit of the intestine. Colon crypts and the surrounding lamina propria house different cell types, including epithelial cells, stem cells, enterocytes, goblet cells, as well as cells of the innate and adaptive immune systems (Clevers, 2013; Mowat and Agace, 2014). Here we describe a technique for the isolation of mouse intestinal crypt cells as well as their characterization by flow cytometry analysis (FACS) (Del Reino et al., 2012).
Keywords: Flow cytometry Colon Intestinal Crypt cell Mouse
Materials and Reagents
Falcon 50 ml conical tubes (VWR International, catalog number: 21008-240 )
Falcon 15 ml conical tubes (VWR International, catalog number: 21008-216 )
10 cm Petri dishes (Corning, Falcon®, catalog number: 353046 )
Gauze, mesh size approximately 100 μm (Tegosa, catalog number: 10018 )
Cell strainers (70 μm) (Corning, Falcon®, catalog number: 352350 )
Cell strainers (40 μm) (Corning, Falcon®, catalog number: 352340 )
25G needle (Premier Healthcare & Hygiene, BD Microlance, catalog number: 300600 )
5 ml syringe (BD, catalog number: 309649)
5 ml round-bottom flow cytometry tube (BD Biosciences, catalog number: 352063 )
8- to 10-week old mice (Mus musculus) (male or female) (we used the C57BL/6 strain)
Hank’s balanced salt solution medium (HBSS) (Life Technologies, Gibco®, catalog number: 2420-091 )
Penicillin-Streptomycin Solution 100x (Biowest, catalog number: L0022 )
Ethylenediaminetetraacetic acid (EDTA) (AppliChem GmbH, Panreac, catalog number: 13102/ 131026 )
Fetal bovine serum (FBS) (Life Technologies, catalog number: GCS0103-500 )
APC-conjugated anti-CD45 antibody (Beckman Coulter, catalog number: 732158 )
FITC-conjugated anti-CD4 antibody (Beckman Coulter, catalog number: 731999 )
PE-conjugated anti-Ly6G antibody (Beckman Coulter, catalog number: 732487 )
Biotinylated anti-F480 antibody (eBioscience, catalog number: 13-4801 )
PeyC7-conjugated anti-CD8 antibody (Biolegend, catalog number: 100721 )
ECD-conjugated streptavidin (Beckman Coulter, catalog number: IM3326 )
Dispase II (Life Technologies, catalog number: 17105-041 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 17105-041”.
Phosphate-buffered saline (PBS) (see Recipes)
Disaggregation medium (see Recipes)
Equipment
Dissection equipment (forceps & scissors)
Scalpel or razor blade
37 °C chamber (Thermo Fisher Scientific, HeraeusTM)
Refrigerated centrifuge (Thermo Fisher Scientific, HeraeusTM, model: MegafugeTM 2.012 )
Neubauer chamber cell counter
Orbital shaking platform (Kühner AG, Lab-Shaker)
CO2 chamber
Cytomics fc500 flow cytometer (Beckman Coulter)
Microscope (ZEISS, model: Axiovert 40CFL )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Alsina-Beauchamp, D., Reino, P. D. and Cuenda, A. (2015). Isolation and Flow-cytometric Analysis of Mouse Intestinal Crypt Cells. Bio-protocol 5(21): e1635. DOI: 10.21769/BioProtoc.1635.
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Category
Cancer Biology > Cancer stem cell > Cell biology assays
Stem Cell > Adult stem cell > Intestinal stem cell
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1,636 | https://bio-protocol.org/exchange/protocoldetail?id=1636&type=0 | # Bio-Protocol Content
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Electroretinogram (ERG) Recordings from Drosophila
Elisheva Rhodes-Mordov*
Hadar Samra*
Baruch Minke
*Contributed equally to this work
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1636 Views: 14644
Edited by: Oneil G. Bhalala
Reviewed by: Masahiro MoritaFabiana Scornik
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor Potential (TRP) channels as its target. The great advantage of using invertebrate photoreceptors is the simplicity of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics. This last feature is mainly attributed to Drosophila melanogaster as a preferred animal model.
The Electroretinogram (ERG) is an extracellular voltage recording from the entire eye, which reflects the total electrical activity arising from the retina in response to a light stimulation. The Drosophila ERG light response is robust and easily obtained, thus making it a convenient method to identify defects in the light response as a result of mutations. The Prolonged Depolarizing Afterpotential (PDA) is a useful ERG phenomenon that can be recorded from white-eyed flies following intense blue light. It is induced by a massive photo-conversion of the photopigment rhodopsin to its dark stable state called metarhodopsin, due to failure of light response termination. Unlike the light coincident ERG recording, which declines quickly to the dark baseline after the cessation of the light stimulus, the PDA response continues long (hours) after light offset. However, this response can be suppressed to the dark baseline at any time by photo-conversion of metarhodopsin back to rhodopsin, by application of an intense orange light stimulus (see Figure 7; Minke, 2012). The PDA has been used as an important tool to screen for visual defective mutant (Minke, 2012).
Keywords: Drosophila photoreceptors Electroretinogram (ERG) Photoresponse Drosophila mutants in vivo electrical recordings
Materials and Reagents
Sterile disposable filter (0.2 μm pore size, aPES membrane, 75 mm diameter) [such as NalgeneTM Rapid-FlowTM Sterile Disposable Filter Units (Thermo Fisher Scientific)]
1 borosilicate glass capillary (OD 1.0 mm, ID 0.58 mm) (Harvard Apparatus)
3 silver filaments (1 mm diameter, 10 cm length) [such as Electrode AG/AGCL 1 mm DIA (WPI)]
1 ml/2 ml Syringe + 1 ml/2 ml Syringe with elongated tip [such as MEDI-PLUS 1ml without needle (KDL Medical Product Company)]
Syringe filter (0.22 μm pore size, suitable for a 4 mm syringe tip) with PVDF membrane [such as Millex-GV Syringe Driven Filter Unit (Merck Millipore Corporation)]
White-eyed Drosophila flies (available for purchase at Bloomington Drosophila Stock Center)
Note: Red-eyed Drosophila flies are also suitable for ERG recordings, but a PDA cannot be induced in these flies.
Homemade low temperature melting wax composed of mixture of paraffin and bee wax
Redux cream for electrocardiography [such as Redux Electrolyte Crème (Parker Laboratories)]
Ringer’s solution (see Recipes)
Equipment
Horizontal Micropipette puller [such as programmable Flaming/Brown type micropipette puller (Sutter Instrument Company, model: P-97 )] with platinum filament. Less expensive pullers [such as Narishige vertical puller (NARISHIGE Group, model: PP-830 )] can also be used
Pulsed gas flow anesthesia system with injector [such as the Sleeper system (Inject+Matic Sleeper)] connected to CO2 tank
Fly stand [such as Alnico Shallow Pot Magnet (Eclipse Magnetics)] (Figure 1, #2, Figure 2, #2 and Video 1)
Fine tweezers [such as tweezers (Biologie, model: number 5 )] and fine paint brush [such as paint brush (Rekab, model: series 40, number 3 )]
Homemade Wax filament heater + soldering iron composed of a platinum-iridium (0.25 mm diameter) filament and a holder (Figure 3)
Dissecting Stereoscopic zoom Microscope [such as Nikon Stereozoom Microscope (Nikon Corporation, model: SMZ-2 )]
“Cold” illuminator [such as KL 1500 LCD (SCHOTT North America, model: KL 1500 LCD )] with heat and red filters [such as KG3 and RG620, respectively (SCHOTT North America, models: KG3 and RG620 )]
Computer with electric signal processor (such as Clampex) and data analysis program (such as Clampfit)
Stereo-microscope with at least 4 different magnification settings [such as the Wild M5 (Leica Microsystems) with 6, 12, 25 and 50 magnification settings] (see Figure 1, #3 and Video 1)
Vibration Isolated Table
Dark Faraday cage covered in black cloth (Figure 1, #6 and Video 1).
On/Off magnet block to provide convenient placement of the fly stand [such as complete Magnetic Base (Eclipse Magnetics, model: E905 )] (Figure 2, #5 and Video 1)
4 triple axis coarse mechanical micromanipulators [such as the Three-Axis Compact Coarse Micromanipulator (Tritech Research, Narishige, model: M-2 )] (Figure 1, #4, Figure 2, #3 and Video 1)
1 triple axis, fine, very stable mechanical micromanipulator for holding the recording electrode [such as the Leitz Mechanical Micromanipulator (Leica Microsystems)] (Figure 1, #8)
Light detector phototransistor for light monitoring (to be fixed on one of the micromanipulators) (Figure 1, #5, Figure 2, #4 and Video 1).
Shutter system [such as LS2 2 mm Uni-stable Shutters (Uniblitz, Vincent Associates®)] + shutter driver [such as VCM-D1 Single Channel Uni-stable (Uniblitz, Vincent Associates®)]
High-pressure ozone-free 75 W Xenon lamp (operating on 50 W) + Schott KG3 heat filter + 2 condenser lenses + fiber optic (3 mm diameter, 1.3 m long) conducting light into the Faraday cage for light stimulation
Lamp power supply (such as PTI LPS-220)
Data acquisition system [such as Digidata 1550 digitizer (Molecular Devices, model: Digidata 1550 digitizer )]
2 glass electrode holders suitable for capillary O.D. 1 mm (Figure 1, #1, Figure 2, #1 and Video 1)
Microelectrode preamplifier system with head-stage impedance tester, at least x10 amplification [such as Extracellular Preamplifier Current Pump System (Dagan, model: 2400A )] (Figure 1, #7 and Video 1)
Pulse generator [such as Master 8 and Master 9 (A.M.P.I, models: Master 8 and Master 9 )]
Neutral Density (ND) filters [such as ND filters (Balzer) and color filters including Red (SCHOTT North America, model: RG610 ), Orange (SCHOTT North America, model: OG590 ) and Blue (SCHOTT North America, model: BG63 )]
Figure 1. An overview of the ERG setup. #1-electrode holder, #2-fly stand, #3- stereo-microscope, #4-micromanipulator, #5-light detector phototransistor, #6- Faraday cage, #7-amplifier head-stage, #8-Leica-Leitz Mechanical Micromanipulator.
Figure 2. A magnified view of the ERG setup. #1-electrode holder, #2-fly stand, #3- micromanipulator, #4-light detector probe, #5-magnet block.
Figure 3. A homemade instrument for immobilizing the living fly. A platinum-iridium (0.25 mm diameter) filament is electrically connected to a power supply with a controlled current output. The filament is attached via insolating plastic to a copper rod holder (heater and soldering iron).
Video 1. A general overview of the ERG set
Procedure
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Copyright: © 2015 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:
Rhodes-Mordov, E., Samra, H. and Minke, B. (2015). Electroretinogram (ERG) Recordings from Drosophila. Bio-protocol 5(21): e1636. DOI: 10.21769/BioProtoc.1636.
Weiss, S., Kohn, E., Dadon, D., Katz, B., Peters, M., Lebendiker, M., Kosloff, M., Colley, N. J. and Minke, B. (2012). Compartmentalization and Ca2+ buffering are essential for prevention of light-induced retinal degeneration. J Neurosci 32(42): 14696-14708.
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Category
Neuroscience > Neuroanatomy and circuitry
Neuroscience > Sensory and motor systems
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1,637 | https://bio-protocol.org/exchange/protocoldetail?id=1637&type=0 | # Bio-Protocol Content
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Peer-reviewed
Computational Identification of MicroRNA-targeted Nucleotide-binding Site-leucine-rich Repeat Genes in Plants
Zhu-Qing Shao*
Yan-Mei Zhang*
Bin Wang
Jian-Qun Chen
*Contributed equally to this work
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1637 Views: 9837
Edited by: Tie Liu
Reviewed by: Alberto Carbonell
Original Research Article:
The authors used this protocol in Sep 2014
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The authors used this protocol in:
Sep 2014
Abstract
Plant genomes harbor dozens to hundreds of nucleotide-binding site-leucine-rich repeat (NBS-LRR, NBS for short) type disease resistance genes (Shao et al., 2014; Zhang et al., 2015). Proper regulation of these genes is important for normal growth of plants by reducing unnecessary fitness costs in the absence of pathogen infection. Recent studies have revealed that microRNAs are involved in regulation of NBS genes in plants (Zhai et al., 2011; Shivaprasad et al., 2012). This protocol describes computational methods for the genome-wide identification of plant NBS genes potentially regulated by microRNAs.
Equipment
Personal computer (an internet connection is needed) (Intel Core i5-2300 CPU, 8 GB RAM)
Sequence data and software
Sequence data compilation
The coding sequence (CDS) and protein sequences of interested genomes should be downloaded from relevant databases. A recommended database containing a relatively large number of sequenced plant genomes is Phytozome (http://www.phytozome.org/) (Goodstein et al., 2012). MicroRNAs of interested genomes can be retrieved from the miRBase (http://www.mirbase.org/) (Kozomara and Griffiths-Jones, 2014) or from in-house sequenced data.
Required software and online tools
The following software should be locally installed in your computer:
Hmmer 3.0 (http://hmmer.janelia.org/) (Johnson et al., 2010), for perform local hidden Markov models (HMM) search of NBS homologous proteins.
NCBI BLAST+ or NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), for perform local blastp search of NBS homologous proteins.
ActivePerl 5.14.2 (http://www.activestate.com/activeperl/downloads), for running scripts written in perl language.
Manuals for installation/operation of these softwares could be downloaded in the referred websites.
The online tools to be used are:
COILS (http://www.ch.embnet.org/software/COILS_form.html) (Lupas et al., 1991), a program for identification of coiled-coils (CC) domain in protein sequences.
Pfam (http://pfam. sanger.ac.uk/) (Finn et al., 2014), a database for identification of protein domains.
psRNATarget (http://plantgrn.noble.org/psRNATarget/) (Dai and Zhao, 2011), a website designed for prediction microRNA targets in plants.
Procedure
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Copyright: © 2015 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:
Shao, Z., Zhang, Y., Wang, B. and Chen, J. (2015). Computational Identification of MicroRNA-targeted Nucleotide-binding Site-leucine-rich Repeat Genes in Plants. Bio-protocol 5(21): e1637. DOI: 10.21769/BioProtoc.1637.
Shao, Z. Q., Zhang, Y. M., Hang, Y. Y., Xue, J. Y., Zhou, G. C., Wu, P., Wu, X. Y., Wu, X. Z., Wang, Q., Wang, B. and Chen, J. Q. (2014). Long-term evolution of nucleotide-binding site-leucine-rich repeat genes: understanding gained from and beyond the legume family. Plant Physiol 166(1): 217-234.
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Category
Plant Science > Plant molecular biology > RNA
Systems Biology > Interactome > Gene network
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1,638 | https://bio-protocol.org/exchange/protocoldetail?id=1638&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells
MS Michael Peter Smith
HY Helen Young
AH Adam Hurlstone
CW Claudia Wellbrock
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1638 Views: 47506
Edited by: HongLok Lung
Reviewed by: Nicoletta Cordani
Original Research Article:
The authors used this protocol in Oct 2014
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The authors used this protocol in:
Oct 2014
Abstract
Understanding how immune cells such as macrophages interact with cancer cells is of increasing interest, as cancer treatments move towards combing both targeted- and immuno-therapies in new treatment regimes. This protocol is using THP-1 cells, a human leukemia monocytic cell line that can be differentiated into macrophages. This allows studying the effects of the macrophage secretome on cancer cells (on e.g., growth, drug response or gene expression) in co-cultures without direct cell contact interactions. This is an important aspect as it removes the presence of any phagocytic aspect to changes in the cancer cell number and behaviour. The in vitro THP-1 monocyte differentiation into polarized macrophages was used to study the effects of both M1 and M2 type populations of macrophages on melanoma cells (Smith et al., 2014; Tsuchiya et al., 1980). M1 type macrophages are classically thought to be tumour suppressing as opposed to M2 type macrophages, which are thought to possess tissue repairing and tumour growth promoting activities.
Keywords: Co-culture Transwell Macrophages THP1 Melanoma
Materials and Reagents
Transwell inserts 0.4 μM (BD Biosciences, catalog number: 353493 )
Note: Currently, it is “Corning, Falcon®, catalog number: 353493 ”.
Sterile 15 ml centrifuge tubes
20 µl and 1 ml tips
Corning® 6 well cell culture plates (Sigma-Aldrich, catalog number: CLS3516-50EA )
THP-1 cells (ATCC, catalog number: ATCC® TIB-202TM )
Foetal bovine serum (500 ml) (heat inactivated by manufacturer) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
Penicillin/Streptomycin soln (100x) stabilised (100 ml) (Sigma-Aldrich, catalog number: P4333 )
Dulbeccos PBS (1x) w/o Ca2+ or Mg2+ (500 ml) (Sigma-Aldrich, catalog number: PBS2541 )
Trypsin-EDTA (1x) (100 ml) (Sigma-Aldrich, catalog number: T3924-100 ml )
EDTA disodium salt AR E (500 gm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: FKCF/0700/53 )
DMEM w/L-Glutamine, Pyruvate & sodium bicarbonate (500 ml) (Sigma-Aldrich, catalog number: D6429-24x )
RPMI 1640 w/L-Glutamine-Bicarbonate (500 ml) (Sigma-Aldrich, catalog number: R8758 )
12-O-tetradecanoylphorbol-l3-acetate (PMA) (Sigma-Aldrich, catalog number: P1585 )
Recombinant human interferon type gamma (IFN-γ) (Peprotech, catalog number: 300-02 )
Recombinant human interleukin 4 (IL-4) (Peprotech, catalog number: 200-04 )
Recombinant human interleukin 13 (IL-13) (Peprotech, catalog number: 200-13 )
Lipopolysaccharides from Escherichia coli (Sigma-Aldrich, catalog number: L2630 )
DMEM/FCS media (see Recipes)
RPMI/FCS media (see Recipes)
IL4 stock solution (see Recipes)
IL13 stock solution (see Recipes)
IFN-γ stock solution (see Recipes)
PMA (see Recipes)
LPS (see Recipes)
Equipment
Benchtop Centrifuge
Aspirator
Haemocytometer
Tissue culture hood
CO2 incubator
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Smith, M. P., Young, H., Hurlstone, A. and Wellbrock, C. (2015). Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells. Bio-protocol 5(21): e1638. DOI: 10.21769/BioProtoc.1638.
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Category
Cancer Biology > Tumor immunology > Cell biology assays
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1,639 | https://bio-protocol.org/exchange/protocoldetail?id=1639&type=0 | # Bio-Protocol Content
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Culture of Megakaryocytes from Human Peripheral Blood Mononuclear Cells
Vishal Salunkhe
Petros Papadopoulos
LG Laura Gutiérrez
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1639 Views: 25294
Edited by: Ralph Bottcher
Reviewed by: Salma Hasan
Original Research Article:
The authors used this protocol in Mar 2013
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Mar 2013
Abstract
Megakaryocytes are the precursor cells of platelets and are bona fide resident cells in the bone marrow but extremely low in numbers (~1% of total nucleated cells). Upon terminal differentiation, megakaryocytes increase their size, become polyploid and develop a demarcation membrane system. Mature megakaryocytes form proplatelets, which are cytoplasmic extensions that protrude through the endothelial cell layer of venous sinusoids within the bone marrow, entering into the blood circulation and, subsequently, releasing platelets. Despite limited in numbers, megakaryocytes have been successfully isolated from bone marrow (Tolhurst et al., 2012), adult peripheral blood (Mazur et al., 1990; Thornton et al., 1999), cord blood (Sun et al., 2004) and also from embryonic stem cells (Pick et al., 2013; Eto et al., 2002). These procedures rely on immunostaining using antibodies against megakaryocyte surface markers (i.e. CD41 or CD42b) to isolate an enriched population of megakaryocytes. Here, we describe a culture method wherein megakaryocytes can be grown and differentiated in vitro from human peripheral blood mononuclear cells (PBMCs) directly without the need of initial isolation of CD34+ cells. This method is based on a previously published culture method of human erythroid progenitor cells from PBMCs (Borg et al., 2010; Leberbauer et al., 2005). Although the purity of megakaryocytes is not 100% in this culture method, an enriched fraction of megakaryocytes can be further isolated using BSA gradient or cell-sorting techniques. In addition, our method offers the possibility to freeze the cultures after minimal expansion of yet undifferentiated megakaryocytes, which will yield equal megakaryocyte cultures after thawing when compared to fresh uninterrupted cultures. As this has been proven difficult with CD34+ sorted pluripotent cells, it allows managing samples and to perform downstream analysis when human material is not always available.
Materials and Reagents
Note: *These materials/reagents can be replaced by similar alternatives.
NUNC tubes-50 ml (Thermo Fisher Scientific, catalog number: 339651 )*
Whole blood (aseptic) in anticoagulant (~ 50 ml volume)
Phosphate buffer saline (PBS) (pH 7.4)
Trisodium Citrate Dihydrate (TNC) buffer (38 gram/L demi water, pH 7.0) (Sigma-Aldrich, catalog number: S1804 )
Percoll® (GE Healthcare, catalog number: 17-0891-09 )
StemSpanTM SFEM (500 ml) (STEMCELL technologies, catalog number: 09650 )
Lipids Cholesterol Rich from adult bovine serum (Sigma-Aldrich, catalog number: L4646 )*
Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P4333 )*
Recombinant human stem cell factor (SCF) (Life technologies, catalog number: PHC2115 )*
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: PHC2115”.
Recombinant human thrombopoietin (TPO) (Life technologies, catalog number: PHC9514 )*
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: PHC9514”.
Recombinant human Erythropoietin (EPO) (R&D Systems, catalog number: 287-TC-500 )*
(Optional) Recombinant Human Flt3-Ligand (FLT3-L) (Peprotech, catalog number: 30019 )*
Culture media (see Recipes)
Basis medium (see Recipes)
Phase I medium (expansion) (see Recipes)
Phase II medium (commitment) (see Recipes)
Phase III medium (differentiation) (see Recipes)
Equipment
Note: *Indicated equipment can be replaced by similar alternatives.
Centrifuge-Rotanta 460 (Hettich, catalog number: 5650 )*
CASY Cell counter (Roche, model: CASY TTC )*
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Salunkhe, V., Papadopoulos, P. and Gutiérrez, L. (2015). Culture of Megakaryocytes from Human Peripheral Blood Mononuclear Cells. Bio-protocol 5(21): e1639. DOI: 10.21769/BioProtoc.1639.
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Category
Cell Biology > Cell isolation and culture > Cell differentiation
Cell Biology > Cell isolation and culture > Cell isolation
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Which buffer can be used to prepare the BSA in different concentration for the density gradient?
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164 | https://bio-protocol.org/exchange/protocoldetail?id=164&type=1 | # Bio-Protocol Content
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Peer-reviewed
Activation of ER-regulated Fusion Proteins In Vivo
JR Jason Reuter
Published: Dec 20, 2011
DOI: 10.21769/BioProtoc.164 Views: 12468
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Abstract
Regulatable protein fusions can be made by attaching the hormone binding domain of the estrogen receptor to the N or C-terminus of a protein of interest. Activation of such ER-fusion proteins in vivo can be achieved by daily administration of a synthetic ligand, 4-hydroxytamoxifen (4OHT), via intraperitoneal (i.p.) injection.
Materials and Reagents
Corn oil (Sigma-Aldrich, catalog number: C8267 )
200-proof ethanol (Sigma-Aldrich, catalog number: E7023 )
4OHT (Sigma-Aldrich, catalog number: H7904 )
Equipment
Sonic dismembrator (Thermo Fisher Scientific, model: 100 )
Centrifuges
Hybridization oven
Procedure
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Reuter, J. (2011). Activation of ER-regulated Fusion Proteins In Vivo. Bio-101: e164. DOI: 10.21769/BioProtoc.164.
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Category
Molecular Biology > Protein > Expression
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1,640 | https://bio-protocol.org/exchange/protocoldetail?id=1640&type=0 | # Bio-Protocol Content
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Peer-reviewed
Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria
SG Sébastien Gélis-Jeanvoine
NB Nienke Buddelmeijer
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1640 Views: 8024
Edited by: Fanglian He
Reviewed by: Soazig Le Guyon
Original Research Article:
The authors used this protocol in Feb 2015
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Feb 2015
Abstract
The topology of membrane proteins and enzymes can be determined using various methods including reporter protein fusions and accessibility of cysteine residues to alkylating agents. Here we describe a variation of the substituted cysteine accessibility method to determine membrane topology and activity of enzymes containing an active site cysteine. Membrane topology of proteins can be predicted using different programs and the actual membrane topology can be determined by monitoring the accessibility of cysteine residues introduced in periplasmic (exposed) or cytoplasmic (not exposed) loops to alkylating agents. A two-step protocol is described where whole Escherichia coli (E. coli) cells are first treated with or without a membrane impermeable thiol reagent (2-sulfonatoethyl)-methane thiosulfonate (MTSES) and subsequently labeled with an alkylating reagent maleimide polyethyleneglycol (malPEG). When cysteine residues are accessible to MTSES, and thus exposed to (or accessible from) the periplasm, their free thiol groups covalently react with MTSES and consequently, are blocked for alkylation with malPEG. The thiol groups of cytoplasmic or membrane-embedded cysteine residues are not accessible to MTSES and proteins can be alkylated with malPEG resulting in an increase in molecular weight of 5 kDa. In the second part of the protocol, accessibility of cysteine residues is used to address the acylation state of enzymes that form stable thioester acyl intermediates. Thioesters can be specifically cleaved by neutral hydroxylamine, leading to a free thiol group of the active site cysteine that can then be alkylated with malPEG.
Materials and Reagents
Escherichia coli K12 cells
Distilled sterile water (dH2O)
Acetone (Sigma-Aldrich, catalog number: 24201-1L-R )
Chloroform (CHCl3) (VWR International, catalog number: 22711.290 )
Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: 27242-500G-R )
Ethanol (EtOH) (100%) (Sigma-Aldrich, catalog number: 24103-5L-R )
Note: Product 24103 has been discontinued.
Tris(hydroxymethyl)aminomethane hydrochloride (Trizma base) (Sigma-Aldrich, catalog number: 337411KG )
1 M KH2PO4 (Merck Millipore Corporation, catalog number: A148973 )
1 M Potassium phosphate dibasic K2HPO4 (Fluka, catalog number: 60353 )
Note: Currently, it is “ Sigma-Aldrich, catalog number: 60353 ”.
Sodium dodecyl sulfate 20%SDS 20% (biosolve-chemicals, catalog number: 19812323 )
Urea (Merck Millipore Corporation, Calbiochem®, catalog number: 66612 )
NaOH (Merck Millipore Corporation, catalog number: 21778425 )
Bromophenol blue (Sigma-Aldrich, catalog number: 114391 )
Glycerol (VWR International, catalog number: 24388.295 )
Parafilm (Thomas Scientific, Pichiney Plastic Packaging, catalog number: PM-992 )
50 mM Phosphate buffer (PB) (pH 7.0) (see Recipes)
250 mM Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich，catalog number: ED2SS ) (see Recipes)
0.3 M Sodium (2-sulfonatoethyl) methane thiosulfonate (MTSES) (Anatrace, catalog number: S110MT ) (see Recipes)
1 M L-cysteine (IGN, catalog number: 194646 ) (see Recipes)
Note: Currently, it is “mpbio, catalog number: 194646 ”.
Methanol (MeOH) (95%) (Sigma-Aldrich, catalog number: 32213-1L ) (see Recipes)
1 M Tris-HCl (see Recipes)
Denaturation buffer (see Recipes)
PEG buffer (see Recipes)
1.2 mM Methoxypolyethylene glycol 5000 maleimide (malPEG) (Sigma-Aldrich, catalog number: 63187 ) (see Recipes)
Hydroxylamine solution (HA) (Sigma-Aldrich, catalog number: 59417 ) (see Recipes)
1 M Tris.HCl, 1% SDS (see Recipes)
SDS-Sample buffer (see Recipes)
DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 ) (see Recipes)
Equipment
Incubator (37 °C) (Infors AG)
Spectrophotometer (600 nm) (Eppendorf, model: Biophotometer plus )
Tabletop centrifuge (Eppendorf, model: 5472R/5427R )
Gelectrophoresis apparatus (Bio-Rad Laboratories, model: Miniprotean II )
Western blot apparatus (Bio-Rad Laboratories, model: Transblot Turbo )
Vortex Genie 2 (Scientific Industries)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Gélis-Jeanvoine, S. and Buddelmeijer, N. (2015). Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria. Bio-protocol 5(21): e1640. DOI: 10.21769/BioProtoc.1640.
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Category
Microbiology > Microbial biochemistry > Protein
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Structure
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1,641 | https://bio-protocol.org/exchange/protocoldetail?id=1641&type=0 | # Bio-Protocol Content
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Peer-reviewed
Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry
KM Kiana Mahdaviani
IB Ilan Benador
OS Orian Shirihai
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1641 Views: 13616
Reviewed by: Hong-guang XiaValentine V Trotter
Original Research Article:
The authors used this protocol in Mar 2014
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Mar 2014
Abstract
Brown adipose tissue (BAT) has the unique ability to dramatically increase mitochondrial uncoupled fuel oxidation for thermogenesis in response to adrenergic stimulation. A key parameter in assessing brown adipocyte thermogenic capacity is mitochondrial uncoupling as determined by respiration. Measuring mitochondrial oxygen consumption rate (OCR) therefore provides valuable information to study the regulation and dysregulation of fuel metabolism and energy expenditure. Adding measurements of mitochondrial membrane potential allows for more in-depth interpretation of the respirometry data. Here we provide protocols for measuring respiration in adherent intact and plasma membrane permeabilized brown adipocytes using the Seahorse XF Analyzer. In the protocol Part I, a combination of norepinephrine and free fatty acids are used to induce uncoupled respiration. The ATP Synthase inhibitor oligomycin, the chemical uncoupler FCCP, and the complex III inhibitor Antimycin A are then used to measure coupled, maximal, and non-mitochondrial oxygen consumption, respectively. In the protocol Part II, the plasma membrane is permeabilized with recombinant perfringolysin O, a cholesterol-dependent cytolysin that oligomerizes into pores exclusively in the plasma membrane. This permits experimental control of metabolite availability without separating mitochondria from the native cell environment.
Part I. Intact brown adipocyte respiration
Materials and Reagents
Note: Rotenone, antimycin, oligomycin, and FCCP are toxic and light sensitive. Wear personal protective equipment when handling and store stocks in the dark.
3-4 weeks-old C57BL6/J mice
Note: Isolate primary pre adipocytes from brown adipose tissue, then differentiate them in an XF-24 well cell culture microplate for 7 days.
XF assay 24‐well cartridge and cell culture microplate (Seahorse Bioscience, catalog numbers: 100850-001 and 100777-004 )
XF assay 24-well cartridge and utility plate
DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 12100-046 )
ewborn calf serum (Sigma-Aldrich, catalog number: N4887 )
HEPES (Corning, catalog number: 25-060-Cl )
L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-164 )
Penicillin-Streptomycin Solution (100x) (Corning, catalog number: 30-002-Cl )
Sodium L-ascorbate (Sigma-Aldrich, catalog number: A4034 )
Rosiglitazone (used as differentiation agent) (Toronto Research Chemicals, catalog number: R693500 )
Insulin from Porcine Pancreas (Sigma-Aldrich, catalog number: I5523 )
XF-assay/base medium (Seahorse Bioscience, Catalog number: 102365-100 )
D-(+)-Glucose (Sigma-Aldrich, catalog numbers: G8270 )
XF Calibrant solution (Seahorse Bioscience, Catalog number: 100840‐000 )
L-(-)Norepinephrine(+)-bitartrate salt monohydrate (Sigma-Aldrich, Catalog number: N5785 )
Free fatty acids (Palmitic acid or Oleate) (Sigma-Aldrich, catalog number: P0500 )
DMSO (Sigma-Aldrich, catalog number: D2650 )
RPMI medium (no glucose) (Thermo Fisher Scientific, GibcoTM, catalog number: 11879-020 )
Fatty‐acid‐free BSA (CalBiochem, catalog number: 126575 )
BAT differentiation media (see Recipes)
Seahorse assay media (SH assay media) (see Recipes)
Free fatty acid conjugated to the BSA (see Recipes)
Norepinephrine (see Recipes)
Oligomycin (Sigma-Aldrich, catalog number: 75371 ) (see Recipes)
Antimycin A from Streptomyces sp. (Sigma-Aldrich, catalog number: A8674 ) (see Recipes)
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 ) (see Recipes)
Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 ) (see Recipes)
Equipment
CO2-free incubator
8% CO2 incubator
XF24 extracellular flux analyzer (Seahorse Bioscience)
Cell culture hood
pH meter
37 °C Water bath
Procedure
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Copyright: © 2015 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:
Mahdaviani, K., Benador, I. and Shirihai, O. (2015). Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry. Bio-protocol 5(21): e1641. DOI: 10.21769/BioProtoc.1641.
Wikstrom, J. D., Mahdaviani, K., Liesa, M., Sereda, S. B., Si, Y., Las, G., Twig, G., Petrovic, N., Zingaretti, C. and Graham, A. (2014). Hormone‐induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure. EMBO J 33(5): 418-436.
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Category
Cell Biology > Cell metabolism > Carbohydrate
Cell Biology > Cell metabolism > Lipid
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1,642 | https://bio-protocol.org/exchange/protocoldetail?id=1642&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation of Murine Adipose Tissue-derived Mesenchymal Stromal Cells (mASCs) and the Analysis of Their Proliferation in vitro
Per Anderson
AC Ana Belén Carrillo-Gálvez
FM Francisco Martín
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1642 Views: 10557
Reviewed by: Agnieszka Pastula
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
Mesenchymal stromal cells (MSCs) are non-hematopoietic, perivascular cells which support hematopoiesis and are thought to participate in tissue repair in vivo. MSCs can be isolated from various tissues and organs and are defined in vitro as plastic adherent cells expressing CD73, CD90, CD105 (human MSCs) or CD29, CD44, sca-1 (murine MSCs) which can differentiate into osteoblasts, adipocytes, chondroblasts and myocytes. MSCs possess potent immunomodulatory and trophic capacities in vitro and in vivo and have thus emerged as a promising treatment of inflammatory/autoimmune diseases. The use of MSCs for human disease relies on the injection of a large number of cells and much effort has been focused on acquiring MSCs with high proliferative capacity. Thus, establishing simple and accurate protocols for measuring MSC proliferation is of importance for both basic and applied research. The current protocol provides details on how to isolate and measure the proliferation of murine MSCs derived from inguinal and/or intraabdominal adipose tissue (mASCs) using the xCELLigence system and CellTiter-Blue reagent (Carrillo-Galvez et al., 2015; Anderson et al., 2013). The protocols described below can also be easily translated to human MSCs.
Materials and Reagents
Scalpel blades No. 24 (VWR International, catalog number: 233-5487 )
Cell strainers (100 μm and 70 μm) (BD Biosciences, catalog numbers: 352360 and 352350 )
Note: Currently, it is “Corning , catalog numbers: 352360 and 352350”.
Black 96-well plate (Greiner Bio-One GmbH, catalog number: 655076 )
16-well E-plate (E-plate 16) (ACEA BIO, catalog number: 06465382001 )
Tissue culture treated Falcon 96-well plates (BD Biosciences, catalog number: 353072 )
Note: Currently, it is “Corning, catalog number: 353072”.
NuncTM Cell Culture Treated EasYFlasksTM (Thermo Fisher Scientific, catalog number: 156499 )
BALB/c (or any strain of choice) 8-12 weeks old male or female mice (Charles River Laboratories)
Hank’s balanced salt solution (HBSS) w/Calcium w/Magnesium (Biowest, catalog number: L0612-500 )
Collagenase Type I (Sigma-Aldrich, catalog number: C0130-100MG )
MesenCultTM Proliferation Kit with MesenPureTM (Mouse) (STEMCELL Technologies, catalog number: 05512 )
Penicillin-Streptomycin 10,000 U/ml (Life Technologies, Gibco, catalog number: 15140-122 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 15140-122”.
Stable Glutamine 200 mM (Biowest, catalog number: X0551-100 )
DPBS w/o Calcium w/o Magnesium (Biowest, catalog number: L0615-500 )
TrypLETM Express Enzyme (1x) (Life Technologies, catalog number: 12604021 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 12604021”.
Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: 10309433 )
Trypan blue (Sigma-Aldrich, catalog number: T8154-20 ml )
CellTiter-Blue reagent (Promega Corporation, catalog number: G8081 )
Collagenase Type I (see Recipes)
Complete MesenCult medium (see Recipes)
Equipment
Sterile stainless steel scissors and forceps
Neubauer cell counting chamber (VWR International, catalog number: 631-1111 )
Cell culture incubator (THERMO, model: HEPA class 100 )
Phase contrast microscope (OLYMPUS, model: CKX41 )
Centrifuge for cells (Eppendorf, model: 5810R )
xCELLigence RTCA station and control unit (lap top) with the RTCA Software (ACEA BIO/ROCHE)
Fluorometro [Glomax multidetection system (Promega Corporation) or equivalent]
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Stem Cell > Adult stem cell > Hematopoietic stem cell
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1,643 | https://bio-protocol.org/exchange/protocoldetail?id=1643&type=0 | # Bio-Protocol Content
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Peer-reviewed
Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae
Gang Li
MM Margarita Marroquin-Guzman
Richard A. Wilson
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1643 Views: 14556
Edited by: Fanglian He
Reviewed by: Arsalan Daudi
Original Research Article:
The authors used this protocol in Oct 2014
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Oct 2014
Abstract
Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed a two-step cross-linking strategy using DSG and formaldehyde-rather than the one-step formaldehyde cross-linking procedure more frequently employed for analyzing direct protein-DNA interactions - in order to better capture the indirect MoJmjC-MoSOD1 DNA interactions in vivo. In addition, we have shown that two-step cross-linking is suitable for ChIP analysis of direct protein-DNA interactions between a GATA transcription factor, Asd4, and its cognate binding site (Marroquin-Guzman and Wilson, 2015). Here, we provide a detailed protocol for chromatin immunoprecipitation, with versatile two-step cross-linking, in M. oryzae.
Materials and Reagents
Oak Ridge Centrifuge Tubes (Thermo Fisher Scientific, NalgenetmTM, catalog number: 3118-0050 )
Miracloth (EMD Millipore Corporation, catalog number: 475855-1R )
Saccharomyces cerevisiae strain XK1-25: MATa trp1 (kindly provided by Dr. Jin-Rong Xu, Dept. of Botany and Plant Pathology, Purdue University)
pHZ126 yeast shuttle vector (kindly provided by Dr. Jin-Rong Xu, Dept. of Botany and Plant Pathology, Purdue University, USA)
QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
Alkali-cation yeast transformation kit (MP Biomedicals, catalog number: 2200-200 )
QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
UltraClean Midi Prep Kit (MO BIO Laboratories, catalog number: 12700-20 )
Zymoprep Yeast Plasmid Mini Prep II Kit (Epigenetics, catalog number: D2004 )
Formaldehyde (Thermo Fisher Scientific, PierceTM, catalog number: 28906 )
DSG (Thermo Fisher Scientific, catalog number: 20593 )
CelLyticTM PN Plant Nuclear Isolation Kit (Sigma-Aldrich, catalog number: CELLYTPN1 )
Nuclei isolation buffer (NIB) (included in the CelLyticTM PN Plant Nuclear Isolation Kit)
Triton X-100 Solution (10% in H2O) (MBL International, catalog number: JM-2104-100 )
Proteinase inhibitor cocktail (Sigma-Aldrich, catalog number: P8215 )
Deoxycholate (Sigma-Aldrich, catalog number: D6750 )
Sodium butyrate (Sigma-Aldrich, catalog number: B5887 )
Sepharose 4B beads (Sigma-Aldrich, catalog number: CL4B200 )
ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich, catalog number: A2220 )
Anti-IgG agarose (Sigma-Aldrich, catalog number: A0919 )
3x FLAG® Peptide (Sigma-Aldrich, catalog number: F4799 )
Glycine (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15527-013 )
Proteinase K (Thermo Fisher Scientific, catalog number: EO0491 )
RNase A (Thermo Fisher Scientific, catalog number: EN0531 )
Wizard PCR Clean-up kit (Promega Corporation, catalog number: A9281 )
Yeast nitrogen base without amino acids (Difco, catalog number: 0919-15 )
Trp DO supplement (Clontech, catalog number: 630413 )
Disuccinimidyl glutarate (DSG) (Thermo Fisher Scientific, catalog number: 20593 )
Lysing Enzymes from Trichoderma harzianum (Sigma-Aldrich, catalog number: L1412 )
Hygromycin B (Thermo Fisher Scientific, GibcoTM, catalog number: 10-687-010 )
SD-Trp solid medium (see Recipes)
SD-Trp liquid solution (see Recipes)
LB agar plates supplemented with 100 μg/ml ampicillin (see Recipes)
Liquid complete medium (see Recipes)
DSG cross-linking buffer (see Recipes)
1x NIB (see Recipes)
NIBA (see Recipes)
Nuclear lysis buffer (see Recipes)
HEPES-buffered saline (HBS) (see Recipes)
0.1 M glycine HCl (pH 3.5) (see Recipes)
Elution buffer (see Recipes)
OM buffer (see Recipes)
ST buffer (see Recipes)
STC buffer (see Recipes)
PTC buffer (see Recipes)
Bottom medium (see Recipes)
Top medium (see Recipes)
Equipment
Benchtop shakers (Eppendorf, catalog number: M1354-0000 )
VirTis VirSonic 50 Ultrasonic Cell Disrupter Disruptor (VirTis company)
Centrifuge (Eppendorf, model: 5415D )
Mini-Tube Rotators (Thermo Fisher Scientific, Fisher Scientific, catalog number: 05-450-127 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Immunodetection
Molecular Biology > DNA > DNA-protein interaction
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1,644 | https://bio-protocol.org/exchange/protocoldetail?id=1644&type=0 | # Bio-Protocol Content
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Capturing the Driving Role of Tumor-host Crosstalk in a Dynamical Model of Tumor Growth
Sebastien Benzekry
AB Afshin Beheshti
PH Philip Hahnfeldt
LH Lynn Hlatky
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1644 Views: 8449
Edited by: Masahiro Morita
Reviewed by: Pia GiovannelliKate Hannan
Original Research Article:
The authors used this protocol in Mar 2015
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Abstract
In 1999, Hahnfeldt et al. proposed a mathematical model for tumor growth as dictated by reciprocal communications between tumor and its associated vasculature, introducing the idea that a tumor is supported by a dynamic, rather than a static, carrying capacity. In this original paper, the carrying capacity was equated with the variable tumor vascular support resulting from the net effect of tumor-derived angiogenesis stimulators and inhibitors. This dynamic carrying capacity model was further abstracted and developed in our recent publication to depict the more general situation where there is an interaction between the tumor and its supportive host tissue; in that case, as a function of host aging (Benzekry et al., 2014). This allowed us to predict a range of host changes that may be occurring with age that impact tumor dynamics. More generally, the basic formalism described here can be (and has been), extended to the therapeutic context using additional optimization criteria (Hahnfeldt et al., 1999). The model depends on three parameters: One for the tumor cell proliferation kinetics, one for the stimulation of the stromal support, and one for its inhibition, as well as two initial conditions. We describe here the numerical method to estimate these parameters from longitudinal tumor volume measurements.
Keywords: Mathematical oncology Tumor growth Parameters estimation
Software
Matlab (The Mathworks Inc) version 12 or later with optimization toolbox (can also be adapted for Scilab: http://www.scilab.org/index.php/en)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Benzekry, S., Beheshti, A., Hahnfeldt, P. and Hlatky, L. (2015). Capturing the Driving Role of Tumor-host Crosstalk in a Dynamical Model of Tumor Growth. Bio-protocol 5(21): e1644. DOI: 10.21769/BioProtoc.1644.
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Category
Cancer Biology > Proliferative signaling > Tumor formation
Cancer Biology > Proliferative signaling > Tumor microenvironment
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1,645 | https://bio-protocol.org/exchange/protocoldetail?id=1645&type=0 | # Bio-Protocol Content
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Skeletal Myogenesis in vitro
WC William C. W. Chen
BP Bruno Péault
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1645 Views: 9677
Reviewed by: Kae-Jiun Chang
Original Research Article:
The authors used this protocol in Feb 2015
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Feb 2015
Abstract
Mature skeletal myofibers are elongated and multinucleated cells. Many stem/progenitor cell types, including committed muscle stem (satellite cells) and progenitor (myoblasts) cells, muscle-derived stem cells, myogenic endothelial cells, and mesenchymal stem/stromal cells, have been shown to exhibit skeletal myogenesis under appropriate inductive conditions. Committed muscle stem/progenitor cells and multipotent stem/progenitor cells which have skeletal myogenic capacity can typically be differentiated into skeletal myofibers in vitro following extended low-serum exposure. Differentiated cells exhibit distinct fiber-like elongated morphology with multiple nuclei and express unique muscle molecular markers indicating myogenesis, including desmin (early) and fast- and/or slow-myosin heavy chain (mature).
Materials and Reagents
Collagen type-I coated plates (sterilized by UV overnight after coating) (the protocol for coating plates is provided by Sigma-Aldrich in the associated product information)
Myogenic cells in sterile conditions (Refer to Chen et al., 2014 and Gharaibeh et al., 2008 for primary human and mouse cell isolation respectively)
Note: For mouse cells, C2C12 cell line (ATCC, catalog number: CRL-1772) may be used as a positive control.
DMEM high-glucose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11995 )
Fetal bovine serum (FBS) (Invitrogen, catalog number: 10437-028 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028”.
Horse serum (HS) (Invitrogen, catalog number: 26050-088 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 26050-088”.
Chicken embryo extract (CEE) (Accurate Chemical & Scientific Corporation, catalog number: MD-004-D )
Penicillin-Streptomycin (P/S) (Invitrogen, catalog number: 15140-122 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
Monoclonal Anti-Desmin antibody produced in mouse (1:100) (Sigma-Aldrich, catalog number: D1033 ), Mouse anti-fast-MyHC (1:250) (Sigma-Aldrich, catalog number: M4276 ) or Monoclonal Anti-Myosin (Skeletal, Slow) antibody produced in mouse (1:250) (Sigma-Aldrich, catalog number: M8421 )
Normal donkey serum (Jackson Immuno Research, catalog number: 017-000-121 )
Alexa 594-conjugated anti-mouse IgG antibody (1:500) (Invitrogen, Molecular Probes, catalog number: A-21203 ) or Alexa 488-conjugated anti-mouse IgG antibody (1:500) (Invitrogen, Molecular Probes, catalog number: A-21202 )
Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog numbers: A-21203 and A-21202”.
4',6-diamidino-2-phenylindole (DAPI) (100 ng/ml, diluted with DPBS from the stock) (Sigma-Aldrich, catalog number: D9542 )
Mouse-on-mouse (M.O.M) Basic kit (staining kit) (Vector Labs, catalog number: BMK-2202 )
Collagen from calf skin (non-sterile) (Sigma-Aldrich, catalog number: C9791 )
Dulbecco’s DPBS without calcium and magnesium (DPBS) (Invitrogen, catalog number: 14190-250 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: A-21203 and A-21202”.
Methanol (Sigma-Aldrich, catalog number: 322415 )
Acetone (Sigma-Aldrich, catalog number: 270725 )
Formalin (10%) (Sigma-Aldrich, catalog number: HT501128 )
Trypsin-EDTA (1x, diluted from the 10x stock with sterile DPBS, no phenol red) (Invitrogen, catalog number: 15400-054 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 15400-054”.
Collagen type-I (Sigma-Aldrich, catalog number: C9791)
Muscle proliferation medium (see Recipes)
Muscle fusion medium (see Recipes)
Equipment
Cell culture incubator
Fluorescence microscope
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, W. C. W. and Péault, B. (2015). Skeletal Myogenesis in vitro. Bio-protocol 5(21): e1645. DOI: 10.21769/BioProtoc.1645.
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Category
Stem Cell > Adult stem cell > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell differentiation
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1,646 | https://bio-protocol.org/exchange/protocoldetail?id=1646&type=0 | # Bio-Protocol Content
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Peer-reviewed
Observation of Chloroplast Movement in Vallisneria
YS Yuuki Sakai
ST Shingo Takagi
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1646 Views: 8851
Edited by: Samik Bhattacharya
Reviewed by: Sam-Geun Kong
Original Research Article:
The authors used this protocol in Jan 2015
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Abstract
Chloroplasts accumulate to weak light and escape from strong light. These light-induced responses have been known from the 19th century (Böhm, 1856). Up to now, many scientists have developed different methods to investigate these dynamic phenomena in a variety of plant species including the model plant Arabidopsis thaliana, a terrestrial dicot (Wada, 2013). Especially, a serial recording to trace the position of individual chloroplast for the analysis of its mode of movement is critical to understand the underlying mechanism. An aquatic monocot Vallisneria (Alismatales: Hydrocharitaceae, Figure 1A) has contributed over a century to such investigation (Senn, 1908; Zurzycki, 1955; Seitz, 1967), because Vallisneria leaves have rectangular parallelepiped-shaped epidermal cells aligned orderly in a monolayer (Figure 1B), providing an excellent experimental system for microscopic studies. Here we describe a protocol for the up-to-date time-lapse imaging procedures to analyze Vallisneria chloroplast movement. Using this and prototype procedures, the relevant photoreceptor systems (Izutani et al., 1990; Dong et al., 1995; Sakai et al., 2015), association with actin cytoskeleton (Dong et al., 1996; Dong et al., 1998; Sakai and Takagi 2005; Sakurai et al., 2005), and regulatory roles of Ca2+ (Sakai et al., 2015) have been strenuously investigated.
Figure 1. Vallisneria plant. A. Whole plant body; B. A bright-field image of adaxial epidermal cells containing a large number of chloroplasts; C. Culture facilities.
Keywords: Live cell imaging Organelle movement Aquatic plants Light-induced responses
Materials and Reagents
Petri dish
Vallisneria plants
Note: Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1C). The plants were grown under 12 h light/12 h dark regime at 20-24 °C.
Vaseline (Wako Pure Chemical Industries, Siyaku, catalog number: 224-00165 )
Potassium Chloride (KCl) (Wako Pure Chemical Industries, Siyaku, catalog number: 163-03545 )
Sodium chloride (NaCl) (Nacalai tesque, catalog number: 31320-05 )
Calcium Nitrate Tetrahydrate [Ca(NO3)2] (Wako Pure Chemical Industries, Siyaku, catalog number: 039-00735 )
Magnesium Nitrate Hexahydrate [Mg(NO3)2] (Wako Pure Chemical Industries, Siyaku, catalog number: 134-00255 )
PIPES (DOJINDO, catalog number: 345-02225 )
Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, Siyaku, catalog number: 197-02125 )
Artificial pond water (APW) (see Recipes)
100x APW mixture (see Recipes)
200 mM PIPES-NaOH (see Recipes)
Equipment
Fluorescent lamps (National, model: FL20S-PG )
Vacuum pump (ULVAC KIKO, model： DTU-20 )
Microscope (OLYMPUS, model: BX50 )
Neutral-density filters (Fujifilm Corporation)
Interference filter (Toshiba, model: KL-55 )
Cut-off filter (Toshiba, model: O-54 )
Quantum sensor (LI-COR Biosciences, model: LI-190SA )
Data logger (LI-COR Biosciences, model: LI-1400 )
Charge-coupled device camera (QImaging, model: RETIGA 2000RV )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant cell biology > Cell structure
Plant Science > Plant physiology > Photosynthesis
Cell Biology > Cell imaging > Live-cell imaging
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1,647 | https://bio-protocol.org/exchange/protocoldetail?id=1647&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves
YS Yuuki Sakai
SI Shin-ichiro Inoue
ST Shingo Takagi
Published: Vol 5, Iss 21, Nov 5, 2015
DOI: 10.21769/BioProtoc.1647 Views: 8207
Edited by: Samik Bhattacharya
Reviewed by: Sam-Geun Kong
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
An aquatic angiosperm Vallisneria (Alismatales: Hydrocharitaceae) has been used as an excellent experimental material over a century to study the light regulation of dynamic intracellular movements including chloroplast redistribution and cytoplasmic streaming (Senn, 1908; Seitz, 1987; Takagi, 1997). However, understanding of the molecular mechanisms lagged behind because of difficulty in applying modern techniques such as gene transformation to this plant. Especially, which kind of photoreceptors function in these intriguing responses has long been an unsolved topic. Recently, genes encoding plant-specific blue-light receptor phototropins were isolated in Vallisneria, for the first time from aquatic plants (Sakai et al., 2015). Phototropins were identified first as the photoreceptor for hypocotyl phototropism in Arabidopsis thaliana, and now known to regulate many responses including chloroplast photorelocation movements in various plant species (Christie, 2007). Phototropins are localized mainly on the plasma membrane and their auto-phosphorylation induced by blue light is the critical step of signal transduction pathway (Sakamoto and Briggs, 2002; Kong et al., 2006; Kong et al., 2013; Inoue et al., 2010). Here we describe a protocol for in vitro protein phosphorylation assay using crude-microsomal and plasma-membrane-enriched fractions of Vallisneria, which enabled us to verify the presence of phototropins and characterize their auto-phosphorylation responses. After these analyses, Sakai et al. (2015) proposed that Vallisneria phototropins mediate the high-intensity-blue-light-induced chloroplast avoidance response.
Keywords: Crude microsomal fraction Plasma-membrane-enriched fraction Light-induced phosphorylation Aquatic plants SDS-PAGE
Materials and Reagents
Commonly-used medical cotton gauze
0.2 ml tube
Vallisneria plants
Note: Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured to be grown over 100 cm in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1A). The plants were grown under 12 h light/12 h dark regime at 20-24 °C.
Dextran (from Leuconostoc ssp., Mr ~500,000) (Fluka, catalog number: 31392 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 31392 ”.
Polyethylene glycol (Sigma-Aldrich, catalog number: P-3640 )
SDS-PAGE
Deionized water
1% Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
50 mM MOPS-KOH (pH7.6) (DOJINDO, catalog number: 343-01805 )
300 mM sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 196-00015 )
10 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) (DOJINDO, catalog number: 342-01314 )
5 mM ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
10 μg/ml dibutylhydroxytoluene (Tokyo Chemical Industry, catalog number: D0228 )
1% casein (Nacalai tesque, catalog number: 073-19 )
5 mM K2S2O5 (Wako Pure Chemical Industries, Siyaku, catalog number: 161-03345 )
1 mM dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 048-29224 )
2.5 mg/ml pepstatin A (Sigma-Aldrich, catalog number: P-4265 )
2.5 mg/ml aprotinin (Sigma-Aldrich, catalog number: A-4529 )
20 mg/ml EDTA-washed Polyvinylpolypyrolidone (Sigma-Aldrich, catalog number: P-6755 )
MOPS-KOH (DOJINDO, catalog number: 345-02225 )
25 mM MgCl2 (Wako Pure Chemical Industries, Siyaku, catalog number: 135-00165 )
250 mM KCl (Wako Pure Chemical Industries, Siyaku, catalog number: 163-03545 )
40% glycerol (Wako Pure Chemical Industries, Siyaku, catalog number: 075-00616 )
20% 2-mercaptoethanol (Sigma-Aldrich, catalog number: A-6365 )
A small amount of bromophenol blue (Wako Pure Chemical Industries, Siyaku, catalog number: 021-02911 )
0.5 M Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: 252859 )
Homogenizing medium (see Recipes)
Buffer A (see Recipes)
Buffer B (see Recipes)
5x phosphorylation buffer (see Recipes)
ATP mixture (see Recipes)
4x SDS buffer (see Recipes)
Equipment
Blade (Lion Office Products Corp., model: L-300 )
Polytron homogenizer (Kinematica AG, model: PT-35/ 2ST”OD” )
Centrifuge
Ultracentrifuge
Teflon homogenizer (10 ml) (Ikemoto Scientific Technology, model: 812-771-04 )
Lighting system (Sugiura Lab, model: FI-150T )
Cut-off filter (KenkoTokina Corporation, model: Y-44 )
Interference filter (KenkoTokina Corporation, model: BP-45 )
Autoradiography (Fujifilm Corporation)
Procedure
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Category
Plant Science > Plant cell biology > Organelle isolation
Biochemistry > Protein > Modification
Cell Biology > Cell signaling > Phosphorylation
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1,648 | https://bio-protocol.org/exchange/protocoldetail?id=1648&type=1 | # Bio-Protocol Content
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Peer-reviewed
Cryopreservation of Human Serum
YR Yael Rosenberg-Hasson
Holden Maecker
Published: Nov 5, 2015
DOI: 10.21769/BioProtoc.1648 Views: 8639
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Abstract
This protocol describes how to collect and aliquot human serum and store at -80 °C for future cytokine/chemokine/protein profile analysis.
Please use personal protective equipment (PPE) as required when handling samples with potential Bloodborne Pathogens.
Keywords: Serum Cryopreservation Collection & Processing Storage Clot activator
Materials and Reagents
Red top tube (BD, catalog numbers: 366431 , 366441 and 366430 )
Cryovials
Patient whole blood sample in red top tube
Clot activator (optional)
Equipment
Freezing box
-80 °C freezer
Biosafety cabinet
Centrifuge
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Rosenberg-Hasson, Y. and Maecker, H. T. (2015). Cryopreservation of Human Serum. Bio-101: e1648. DOI: 10.21769/BioProtoc.1648.
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Category
Immunology > Immune cell isolation > Maintenance and differentiation
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1,649 | https://bio-protocol.org/exchange/protocoldetail?id=1649&type=0 | # Bio-Protocol Content
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Measurement of H+ Flux in Rice by Non-invasive Micro-test Technology
XZ Xi Zhang
XZ Xiu-Yue Zhang
LY Li-Ping Yin
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1649 Views: 9066
Edited by: Zhaohui Liu
Reviewed by: Wenrong HeCindy Ast
Original Research Article:
The authors used this protocol in Apr 2015
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Apr 2015
Abstract
Rice plants release proton (H+) from root cells into rhizosphere area leading to the acidification of the rhizosphere and increased solubility of ferric iron complexes on the cell membrane, which is important for iron uptakes. Here, we present a detailed protocol to measure H+ flux in root hairs of transgenic rice seedlings and transgenic rice protoplasts by the Non-invasive Micro-test Technique (NMT). The NMT system is based on a non-invasive microelectrode technology that is automatically controlled by a computer, to achieve a three-dimensional, real-time, dynamic characterization of the concentration, velocity, and direction of a variety of molecules or ions. Because there is no need to directly contact the measured cells that could cause cell damage, we are able to obtain accurate and real time information on ion concentration. This is the first protocol that describes the non-invasive micro measurement technique of both root hairs and protoplasts in rice. In NMT, voltage differences are measured at two excursion points that are manipulated using a computer. Voltage differences can be converted into H+ fluxes using the ASET 2.0 (The imFlux® software) and JCal v3.2.1 Software. Analysis of the H+ fluxes provides a simultaneous measure of the crossing of a localized region of the root surface in response to stress, which provides real-time in-situ detection of net ion transport across membranes. This method will promote use of NMT in plant biology.
Keywords: Proton flux Non-invasive Micro-test Technique (NMT) Rice Microelectrode
Materials and Reagents
Cell strainer (400 mesh)
Centrifuge tube (1.5 ml and 10 ml) (Corning Inc., catalog number: MC-150-C )
Round Petri dish (35 mm in diameter) (Corning Inc.)
Square Petri dish (100 mm, pym-100-10) (Xinshengke)
Plastic strips
Constructed vector (Li et al., 2015)
Transgenic Secretory 24 (OsSEC24) (Li et al., 2015) rice seedlings and plasma membrane (PM) ATPase 2 (OsPMA2)-mCherry (Li et al., 2015) plus OsSEC24-GFP double-transgenic rice protoplasts
Cellulase (Onozuka, catalog number: 130419-01 )
Macerozyme (Japan) (catalog number: 121207-01)
M519 (catalog number: 14C0519105B)
Mannitol
MES (pH 5.7)
KCl
NaCl
CaCl2
MgCl2
Sucrose
1%-1.5% agar
Calibration medium buffer (pH 7.0) for NMT (see Recipes)
Calibration medium buffer (pH 5.0) for NMT (see Recipes)
Incubation solution (see Recipes)
Mmg solution (see Recipes)
MS medium (see Recipes)
NT medium (see Recipes)
Protoplasting enzyme solution (see Recipes)
Polyethylene glycol (PEG)-calcium solution (see Recipes)
Standard medium buffer (pH 6.0) for NMT (see Recipes)
Washing buffer (see Recipes)
Equipment
Centrifuge (SIGMA Laborzentrifugen GmbH, model: 3K15 )
Glass beaker (100 ml) (Bomex)
Growth chamber
Liquid ion exchanger (LIX) Holder (YoungerChina)
Microelectrodes (1-2 μm, 4-5 μm) (YoungerUSA)
Micropipettor (100-1,000 μl and 10-100 μl) (Eppendorf)
Non-invasive Micro-test System (YoungerUSA, model: NMT100 series )
Shaker (Focus Technology Co., Shanghai Meditry Instrument, model: THZ-C-1 )
Software
NIS-Elements AR software (available at http://www.youngerusa.com)
ASET 2.0 software (The imFlux® software) (available at http://www.youngerusa.com)
JCal v3.2.1 (available at http://www.youngerusa.com)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhang, X., Zhang, X. and Yin, L. (2015). Measurement of H+ Flux in Rice by Non-invasive Micro-test Technology. Bio-protocol 5(23): e1649. DOI: 10.21769/BioProtoc.1649.
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Category
Plant Science > Plant physiology > Ion analysis
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165 | https://bio-protocol.org/exchange/protocoldetail?id=165&type=1 | # Bio-Protocol Content
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Isolation of Primary Human Keratinocytes from Foreskin
JR Jason Reuter
Published: Dec 20, 2011
DOI: 10.21769/BioProtoc.165 Views: 16165
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Abstract
Keratinocytes are the primary constituents of human skin, the functional barrier between our bodies and the external environment. The balance between keratinocyte differentiation and self-renewal is crucial to skin homeostasis. Primary keratinocyte culture serves as a tractable model for understanding human epithelial cell differentiation as well as self-renewal.
Materials and Reagents
Foreskin
0.05% Trypsin (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
2x dispase (Life Technologies, InvitrogenTM, catalog number: 17105-041 )
DMEM (Life Technologies, InvitrogenTM, catalog number: 11965-092 )
Fetal bovine serum (FBS) (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
Defined keratinocyte serum-free medium (Life Technologies, InvitrogenTM, catalog number: 10744-019 )
154 medium (Life Technologies, InvitrogenTM, catalog number: M-154-500 )
Penicillin and streptomycin (Life Technologies, InvitrogenTM, catalog number: 10378-016 )
Phosphate buffered saline (PBS)
DMEM cultural medium (see Recipes)
50: 50 medium (see Recipes)
Equipment
Centrifuges
Incubator (37 °C and 5% CO2)
Sterile petridish
Cell culture hood
Cell culture dishes
Scissors
Forceps
50 ml falcon tube
Disposable sterile cell culture strainer
Procedure
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Developmental Biology > Cell growth and fate
Cell Biology > Cell isolation and culture > Cell isolation
Cell Biology > Cell isolation and culture > Cell differentiation
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1,650 | https://bio-protocol.org/exchange/protocoldetail?id=1650&type=0 | # Bio-Protocol Content
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13C Tracer Studies of Metabolism in Mouse Tumor Xenografts
AL Andrew N. Lane
JY Jun Yan
TF Teresa W-M. Fan
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1650 Views: 11364
Edited by: Masahiro Morita
Reviewed by: Shannon RuppertYong Teng
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
Mice are widely used for human tumor xenograft studies of cancer development and drug efficacy and toxicity. Stable isotope tracing coupled with metabolomic analysis is an emerging approach for assaying metabolic network activity. In mouse models there are several routes of tracer introduction, which have particular advantages and disadvantages that depend on the model and the questions addressed. This protocol describes the bolus i.v. route via repeated tail vein injections of solutions of stable isotope enriched tracers including 13C6-glucose and 13C5,15N2-glutamine. Repeated injections give higher enrichments and over longer labeling periods than a single bolus. Multiple injections of glutamine are necessary to achieve adequate enrichment in engrafted tumors.
Keywords: SIRM Mouse PDX model NOD/SCID/Gamma mouse Isotopomer distribution analysis
Materials and Reagents
0.5 ml K2-EDTA collection tubes (BD, catalog number: 363706 )
Sterile syringes and gauge 30 needles (Thermo Fisher Scientific, catalog number: 10142534 )
5-inch squares Al foil
Indelible marker pens (black)-Sharpie fine tip
Pasteur pipet
Lancets (MEDIpoint, Inc., model: Goldenrod 5 mm )
Mice bearing tumor xenograft
Note: The strain of immunocompromised recipient mouse depends on type of implant. Young NOD/SCID Gamma (NSG) mice are especially favorable for the patient derived xenograft (PDX) model.
Ketamine for anesthesia if needed, Schedule III controlled substance (Sigma-Aldrich, catalog number: K2753 )
Tracer [e.g., 13C6-glucose, 13C2-1, 2-glucose (60 mg/mouse), or 13C5, 15N2-glutamine (22 mg/mouse)]
Sources
D-Glucose-13C6 (Cambridge Isotope Laboratories, Inc., catalog number: CLM-1396-CTM )
D-Glucose-1, 2-13C2 (Cambridge Isotope Laboratories, Inc., catalog number: CLM-504 )
L-Glutamine-13C5, 15N2 (Cambridge Isotope Laboratories, Inc., catalog number: CNLM-1275 )
Or
D-Glucose-13C6 (Sigma-Aldrich, catalog number: 660663 )
D-Glucose-1, 2-13C2 (Sigma-Aldrich, catalog number: 661422 )
L-Glutamine-13C5, 15N2 (Sigma-Aldrich, catalog number: 607983 )
Liquid nitrogen
70% ethanol
10% Neutral buffered formalin (Thermo Fisher Scientific, ProtocolTM, catalog number: 032-059 )
25% w/v sterile filtered (0.2 µm) 13C glucose in PBS
Note: Stock solution can be frozen and stored at -25 °C.
Sterile wipes (Uline, catalog number: S-16183 )
Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-1 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate dibasic (Na2HPO4) anhydrous (Sigma-Aldrich, catalog number: S-0876 )
Potassium phosphate monobasic (KH2PO4) anhydrous (Sigma-Aldrich, catalog number: P9791 )
10x Phosphate Buffered Saline (PBS) (see Recipes)
Stock solution for 0.2% 13C6-glucose (see Recipes)
Stock solution for 13C5, 15N2-Gln (10 ml) (see Recipes)
Equipment
Mouse restraining system (Plas-Labs, Inc., model: 541-RR )
FirstHand Surgical Instrument Kits for Mice and Rats (Kent Scientific Corporation, model: INSMOUSEKIT )
Blue wax dissection tray (VWR International, catalog number: 10060-188 )
Warming blanket (Kent Scientific Corporation, model: DCT-15 )
Water bottle with DI water (Thermo Fisher Scientific, catalog number: 02-897-11 )
Polystyrene Weigh boats (USA Scientific Inc., catalog number: 2347-1427 )
Calipers for measuring tumor size (Thermo Fisher Scientific, catalog number: 15-077-957 )
2-place Balance for weighing tissues (VWR International, Adventurer Ohaus®, catalog number: 10153-744 )
Refrigerated Microfuge (Eppendorf, model: 5417R with rotor)
Dewar/bucket (Nalgene plastic dewar) for lN2 (Thermo Fisher Scientific, catalog number: S34074C )
Large Styrofoam box
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cancer Biology > Cellular energetics > Tumor microenvironment
Biochemistry > Carbohydrate > Glucose
Cell Biology > Cell metabolism > Carbohydrate
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1,651 | https://bio-protocol.org/exchange/protocoldetail?id=1651&type=0 | # Bio-Protocol Content
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Peer-reviewed
Expression, Purification and in vitro Enzyme Activity Assay of Plant Derived GTPase
AG Annemarie Glöckner
CV Christian A. Voigt
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1651 Views: 9769
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Jul 2014
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Jul 2014
Abstract
Based on gene expression data after biotic stress, the GTPase RabA4c has been suggested to regulate pathogen-induced callose biosynthesis in the model organism Arabidopsis thaliana. We studied the function of RabA4c in its native and dominant negative (dn) isoform. In planta, RabA4c overexpression prevented penetration of the virulent powdery mildew Golovinomyces cichoracearum into epidermal leaf cells. This penetration resistance was caused by enhanced callose deposition at sites of attempted fungal penetration at early time points of infection. By contrast, RabA4c (dn) overexpression did not increase callose deposition or penetration resistance.
In this protocol, we describe the expression, purification and activity assay of the heterologously expressed GTPase RabA4c from A. thaliana based on the publication Ellinger et al. (2014). We fused RabA4c to the fluorophore mCitrine and expressed this protein in the yeast strain Pichia pastoris GS115. For purification of RabA4c, we used the GFP-Trap_A kit (Chromo Tek) which specifically binds to GFP derivatives like mCitrine. The enzyme activity assay was done by using the GTPase Assay Kit from Innova Biosciences. In general, we followed the instructions made by the manufacturers.
Materials and Reagents
Protein expression
pGAPZ A, B, & C Pichia pastoris Expression Vectors (Life Technologies, catalog number: V200-20 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: V200-20”.
ZeocinTM (InvivoGen, catalog number: ant-zn-5b ) for selection
1 % yeast extract (Carl Roth GmbH + Co., catalog number: 2363.3 )
2 % peptone (BD, catalog number: 211677 )
D(+)-Glucose (Carl Roth GmbH + Co., catalog number: X997.3 )
Yeast Peptone Dextrose (YPD) media (see Recipes)
Protein purification
Acid washed glass beads (Sigma-Aldrich, catalog number: G 8772 )
GFP-Trap® (ChromoTek GmbH, catalog number: gta-20 )
NanoOrange Protein Quantification Kit (Invitrogen, catalog number: N-6666 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: N-6666”.
10 mM Tris (pH 7.5)
50 mM NaCl
0.5 mM EDTA (pH 8.0)
100 mM Glycine (pH 2.5)
1 M Tris (pH 10.4)
Dilution buffer (see Recipes)
Wash buffer I (see Recipes)
Wash buffer II (see Recipes)
Elution buffer (see Recipes)
GTPase activity assay
GTPase Assay Kit (Innova Biosciences Ltd., catalog number: 602-0121 )
Negative control: expressed and purified GFP using P. pastoris as expression system (plasmid pGAPZ::eGFP)
Equipment
300 ml flasks
Shaker (160 rpm)
Centrifuge for 50 ml Falcon tubes (3,220 x g)
Vortex
Tube rotator at 4 °C and room temperature
Plate Reader (BioTek Instruments, model: Synergy HTX Multi-Mode Reader)
Tabletop centrifuge (cooling)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant biochemistry > Protein
Plant Science > Plant biochemistry > Protein
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1,652 | https://bio-protocol.org/exchange/protocoldetail?id=1652&type=0 | # Bio-Protocol Content
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Quantifying the Permeability of the Apoplastic Water Barrier in Cosmos Petals
RJ Reinhard Jetter
CB Christopher Buschhaus
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1652 Views: 6936
Edited by: Tie Liu
Reviewed by: Arsalan Daudi
Original Research Article:
The authors used this protocol in Jan 2015
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Abstract
The capacity of plants to minimize uncontrolled water loss is essential for survival in adverse and changing climatic conditions. In order to assess and compare the effectiveness of apoplastic barriers to water, the permeability of the barrier must first be quantified. Studies have accomplished this directly by quantifying tritium flux or indirectly by measuring the influx/efflux of water surrogates such as dyes, chlorophyll, and herbicides. Other studies have relied on comparative methods such as survival rates after drought. These methods rely on radioactive material, correlations, or qualitative comparisons. However, a quantitative method is necessary that directly measures water efflux and that allows easy comparisons within and between experiments, plant parts, plant species, and especially research laboratories. Here we outline in detail a gravimetric protocol first described by Schönherr and Lendzian (1981) that can be set up in less than half a day and completed in one to ten days depending on the plant barrier. This approach has been used in numerous studies on leaf and fruit cuticles and recently also on petals from cosmos (Cosmos bipinnatus; Buschhaus et al., 2015).
Materials and Reagents
Leaf punch (12 millimeter diameter)
Syringe (10 ml)
Syringe (1 ml) with needle
Pipette tip (100 µl)
Pencil (unused)
Sand paper (80 grit) (other grits may work but the author has not tested them)
Tape (3M, Scotch®)
Silica desiccant (Sorbsil Chameleon, BDH Prolabo)
Plant material with minimum dimensions of 13 x 13 millimeters (e.g., ray petals 3-days post-anthesis from Cosmos bipinnatus cv. sensation pinkie; Seeds were obtained from Stokes Seeds)
Silicon grease (Dow Corning Corporation, catalog number: Z273554 )
De-ionized water
Fertilizer (e.g., MiracleGro)
Equipment
Cylinders (machined in-house with an 11 mm diameter opening and an internal capacity of at least 1 ml; Figure 1. Further details are provided in the ‘Notes’ section)
Metal meshing (10 cm x 20 cm x 0.2 cm; 0.7 cm hole diameter; 0.5 holes cm-2)
Plastic box with tight fitting lid (10 cm x 20 cm)
Incubator (Thermo Fisher Scientific, model: Isotemp 210 )
Balance (Sartorius AG, model: CP1245 )
Computer
Software
Spreadsheet software (e.g., Microsoft Excel)
Data logger program to transfer balance data directly to computer (optional)
Procedure
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Category
Plant Science > Plant physiology > Abiotic stress
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1,653 | https://bio-protocol.org/exchange/protocoldetail?id=1653&type=0 | # Bio-Protocol Content
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An Assay to Test the Capacity of Arabidopsis Plant Defensin Type1 Protein to Induce Cellular Zinc (Zn) Tolerance in Yeast
ZS Zaigham Shahzad
LM Laurence Marquès
PB Pierre Berthomieu
FG Françoise Gosti
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1653 Views: 7694
Edited by: Tie Liu
Reviewed by: De Michele Roberto
Original Research Article:
The authors used this protocol in Nov 2013
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Nov 2013
Abstract
Heterologous expression of genes in budding yeast Saccharomyces cerevisiae (S. cerevisiae) is especially suitable to functionally study the corresponding encoded protein at the cellular level (Bonneaud et al., 1991). This is mainly because many strains defective in specific activities are available and could be complemented by homologous genes existing across the eukaryotic kingdom (http://www.yeastgenome.org/). However, the protocol we describe here is not a complementation but a “gain-of-function” assay. It is based on a drop-test assay that we have set up to assess the cellular zinc tolerance conferred by the expression of heterologous genes in the wild-type S. cerevisiae. Different dilutions of a yeast culture expressing the heterologous gene of interest are grown on a range of zinc-enriched plates, and are then compared to the control yeast expressing the empty vector. Working with different concentrations of both yeast and zinc are essential to succeed in describing zinc tolerance phenotype upon yeast transformation (Mirouze et al., 2006). This test has also proven to be valuable to differentiate among related members of gene families as exemplified for Arabidopsis Plant Defensin type1 (Shahzad et al., 2013).
Keywords: Plant defensin Abiotic stress Zinc tolerance Yeast
Materials and Reagents
Plastic foil (Saran Wrap)
Sterile square 12 cm x 12 cm Petri dishes (CML HealthCare, catalog number: BP 120SJ )
Sterile-R Filtropur S (0.2 µm) (SARSTEDT AG, catalog number: 83.1826.001 )
20 ml sterile syringe without needle (Medline Scientific, catalog number: BS-20ES )
50 ml sterile tubes (SARSTEDT AG, catalog number: 62.547.254 )
Autoclaved 1.5 ml Eppendorf tubes (SARSTEDT AG, catalog number: 72.690 )
Sterile inoculating loops (Greiner Bio One International GmbH, catalog number: 731175 )
Sterile pipet tips
Parental BY4741 S. cerevisiae strain (Winston et al., 1995; Brachmann et al., 1998) having the genotypes MATa, his3∆1, leu2∆0, met15∆0, ura3∆0) expressing: the pFL38H vector (Bonneaud et al., 1991), complementing the his3∆1 auxotrophy
Transformed BY4741 yeast harbouring both: the pFL38H vector, complementing the his3∆1 auxotrophy and the pYX212 vector (Ingenius, R&D systems) complementing the ura3∆0 mutation and expressing the entire coding sequences of Arabidopsis PDF1s, i.e., including the signal peptide (Shahzad et al., 2013)
Yeast nitrogen base (YNB) without amino acids and without ammonium sulfate (BD, Difco, catalog number: 233520 )
Ammonium nitrate (NH4NO3) (Sigma-Aldrich, catalog number: A9642 )
D(+)Glucose anhydrous (C6H12O6) (EUROMEDEX, catalog number: UG3050 )
Succinic acid (C4H6O4) (Sigma-Aldrich, catalog number: S7501 )
Potassium hydroxide (KOH) (Bio Basic Canada Inc., catalog number: PB0441 )
Agarose type D-5 (EUROMEDEX, catalog number: D5 )
Zinc sulphate heptahydrate (ZnSO4.7H2O) (Sigma-Aldrich, catalog number: Z4750 )
L-leucine (C6H13NO2) (Sigma-Aldrich, catalog number: L8912 )
L-methionine (C5H11NO2S) (Sigma-Aldrich, catalog number: M9625 )
Sterile ultra-pure water
0.5 M zinc sulfate
10 N KOH (see Recipes)
0.075 M L-leucine (see Recipes)
0.1 M L-methionine (see Recipes)
Sterile liquid and solid selective yeast nitrogen base (YNB) media (see Recipes)
Equipment
Sterile 250 ml borosilicate glass Erlen flask
1,000 ml bottle
Typical 20 µl pipet
Autoclave
Heating oven (Universal Memmert, model: 400 )
New BrunswickTM Agitator (Eppendorf, model: Innova 44 )
A normal cell density meter (Thermo Fisher Scientific, model: 40 )
Laminar flow hood (ADS, model: VP120 )
Centrifuge (Eppendorf, model: 5804R )
Integral water purification system (Merck Millipore Corporation, model: Milli-Q+ )
Scanner (Epson, model: 124OU )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Heterologous expression system > Saccharomyces cerevisiae
Plant Science > Plant physiology > Ion analysis
Cell Biology > Cell-based analysis > Ion analysis
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1,654 | https://bio-protocol.org/exchange/protocoldetail?id=1654&type=0 | # Bio-Protocol Content
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Arabidopsis Leaf Explant Culture
Jing-Jing Wang
Lisha Zhang
Hui-Shan Guo
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1654 Views: 11845
Edited by: Tie Liu
Reviewed by: Teresa Lenser
Original Research Article:
The authors used this protocol in Mar 2015
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Mar 2015
Abstract
In this protocol, Arabidopsis leaf explant culture is described using an adaptation of a previous method (Hu et al., 2000). Cells from the cut edges of leaf explant are able to proliferate and subsequently form calli on the callus induction medium, in which is supplemented with 2,4-D and 6-benzyl aminopurine [6-BA]. 2,4-D, one of the artificial auxin, is able to promote cell mitosis at low concentration. 6-BA, the first generation of synthetic cytokinin, plays an important role in plant cell division. 2,4-D in combination with 6-BA can effectively induce callus formation (Rashmi and Trivedi, 2014). The aim of this protocol is to analyze cell division competence of Arabidopsis plants with different genotypes. This protocol can be modified and applied to culture explants from other types of plant tissues, such as root and stem.
Keywords: Leaf explant Tissue culture Callus
Materials and Reagents
Plastic Petri dish
Parafilm
Millex-GP Filter Unit 0.22 μm (Merck Millipore Corporation, model: R4AA41572 )
2-3 weeks old sterile Arabidopsis plants (Murashige & Skoog solid medium grown)
Murashige & Skoog medium (Duchefa Biochemie, catalog number: P11293.01 )
Phytagel (Sigma-Aldrich, catalog number: MFCD00131909 )
NaOH
Sucrose
2, 4-dichlorophenoxyacetic acid (2, 4-D) (Sigma-Aldrich, catalog number: D7299 ) (see Recipes)
6-benzylaminopurine (6-BA) (Sigma-Aldrich, catalog number: 83488 ) (see Recipes)
Murashige & Skoog solid medium (see Recipes)
Callus induction medium (see Recipes)
Equipment
Arabidopsis growth chamber
Magnetic stirrer and stirring bar
pH meter
Scissors
Tweezers
Flow cabinet
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wang, J., Zhang, L. and Guo, H. (2015). Arabidopsis Leaf Explant Culture. Bio-protocol 5(22): e1654. DOI: 10.21769/BioProtoc.1654.
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Category
Plant Science > Plant physiology > Plant growth
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1,655 | https://bio-protocol.org/exchange/protocoldetail?id=1655&type=0 | # Bio-Protocol Content
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Peer-reviewed
Locomotor Coordination Assay in Rats
J. Regino Perez-Polo
HR Harriet C. Rea
SI Smitha K. Infante
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1655 Views: 8406
Edited by: Soyun Kim
Reviewed by: Tifany Desprez
Original Research Article:
The authors used this protocol in Apr 2015
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Apr 2015
Abstract
Neonatal hypoxia-ischemia (HI) affects 60% of low birth weight infants and up to 40% of preterm births. Cell death and brain injury after HI have been shown to cause long-lasting neurological deficits. Two motor coordination tests on rats that had been exposed to HI on postnatal day 7 (P7) showed that HI in the P7 rat is associated with significant motor coordination impairment. These results call attention to the risks associated with perinatal ischemia and the need for proper treatment to reverse HI-induced deleterious effects.
Keywords: Motor coordination Balance Inclined plane Rat Hypoxia
Materials and Reagents
Rats
Equipment
Note: Both rod and incline wire mesh are self-made from supplies bought at hardware store.
Rod (30.0 cm L x 1.0 cm)
Wire mesh (69.5 cm W x 45 cm L with 3 cm gaps)
Procedure
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How to cite：Perez-Polo, J. R., Rea, H. C. and Infante, S. K. (2015). Locomotor Coordination Assay in Rats. Bio-protocol 5(22): e1655. DOI: 10.21769/BioProtoc.1655.
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Category
Neuroscience > Behavioral neuroscience > Animal model
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1,656 | https://bio-protocol.org/exchange/protocoldetail?id=1656&type=0 | # Bio-Protocol Content
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Telomere Dysfunction Induced Foci (TIF) Analysis
Ilgen Mender
Jerry W. Shay
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1656 Views: 11620
Edited by: HongLok Lung
Reviewed by: Vanesa Olivares-Illana
Original Research Article:
The authors used this protocol in Jan 2015
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Abstract
Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al., 2003).
The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX, and telomeres using an antibody against one of the shelterin proteins such as TRF2 (Takai et al., 2003; de Lange, 2002; Karlseder et al., 1999). The method we describe here can be used in normal human and cancer cells.
Other commonly used methods-Telomere Restriction Fragment (TRF) Analysis (Mender and Shay, 2015b) and Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015a)- in telomere biology can be found by clicking on the indicated links.
Keywords: Telomere dysfunction induced foci Telomeres Telomerase Telomeric DNA damage
Materials and Reagents
Lab-Tek II Chamber slides (Thomas Scientific, catalog number: 154461 )
Cancer or normal human cells
Triton X-100 (J.T. Baker Chemical Co., catalog number: 7-x198 )
Octyl Phenol Ethoxylate (Avantor Performance Materials, J.T. Baker, catalog number: X198-07 )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
Nonidet-P40 (Fluka BioChemika, catalog number: 74385 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 74385”.
Fish gelatin blocking buffer, 10% (Amresco, catalog number: M319 )
Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P )
Anti-phospho-histone H2A.X (Ser139, mouse) Antibody (Merck Millipore Corporation, catalog number: 05-636 )
Anti-TRF2 Antibody [EPR3517 (Ouellette et al., 2000)] (rabbit) (Abcam, catalog number: ab108997 )
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Invitrogen, catalog number: A-21206 )
Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: A-21206”.
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 568 conjugate (Invitrogen, catalog number: A-11004 )
Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: A-11004”.
Vectashield® Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200 )
Deltavision® Immersion Oil N=1.518 (GEHC, part no: 290291717 )
Phosphate-buffered saline (PBS)
MilliQ® water
10x PBS buffer-phosphate buffer saline (see Recipes)
1x PBST buffer (see Recipes)
Equipment
Personal DeltaVision wide-field fluorescent microscope (GE Healthcare, model: PD11435 )
Lamp (Xenon)
Camera (Photometrics)
60x/1.42 N.A. (numerical aperture) objective (Olympus)
Software
DeltaVision® SoftWoRx software (GE Healthcare)
Autoquant® Software (Media Cybernetics)
Imaris® Software (Bitplane Imaris)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mender, I. and Shay, J. W. (2015). Telomere Dysfunction Induced Foci (TIF) Analysis. Bio-protocol 5(22): e1656. DOI: 10.21769/BioProtoc.1656.
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Category
Cancer Biology > Replicative immortality > Cell biology assays
Cancer Biology > Replicative immortality > Cell biology assays
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1,657 | https://bio-protocol.org/exchange/protocoldetail?id=1657&type=0 | # Bio-Protocol Content
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Peer-reviewed
Telomerase Repeated Amplification Protocol (TRAP)
Ilgen Mender
Jerry W. Shay
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1657 Views: 20739
Edited by: HongLok Lung
Reviewed by: Vanesa Olivares-Illana
Original Research Article:
The authors used this protocol in Jan 2015
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Abstract
Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al., 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase- consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC- counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP).
The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al., 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non-telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is analyzed by electrophoresis. TSNT is, an internal standard control, amplified by TS primer. NT is its own reverse primer, which is not a substrate for telomerase. These primers are used to identify false-negative results by if the gel lacks internal control bands.
Keywords: Replicative senescence Telomeres Cancer Stem cells PCR
Materials and Reagents
Cancer cells (H1299 non-small cell lung, A549 non-small cell lung cancer cells)
Tris-HCl (pH 8.3 and pH 8.0)
Magnesium Chloride (MgCl2) (Thermo Fisher Scientific, catalog number: BP241 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
TweenTM 20 (Thermo Fisher Scientific, catalog number: BP337 )
Ethylene glycol-bis(2-aminoethylether)-N, N, N′, N′-tetraacetic acid EGTA (Sigma-Aldrich, catalog number: E3889 )
Ethylenediaminetetraacetic Acid (EDTA) (Thermo Fisher Scientific, catalog number: BP-120 )
Nonidet-P40 (Fluka BioChemika, catalog number: 74385 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 74385 ”.
Glycerol (Sigma-Aldrich, catalog number: G5516 )
2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) (Sigma-Aldrich, catalog number: A8456 )
dNTP (Invitrogen)
Cy5-TS primer (Sigma-Aldrich)
ACX (reverse primer) (Sigma-Aldrich)
TSNT (36-bp internal standard control) (Integrated DNA Technologies)
NT (reverse primer for internal standard) (Sigma-Aldrich)
Takara Taq DNA polymerase, hot start version (Takara Bio Company, ClonTech, catalog number: R007A )
40% acrylamide and bis-acrylamide solution (19:1) (Bio-Rad Laboratories, catalog number: 161-0144 )
Ammonium persulfate (APS) (Bio-Rad Laboratories, catalog number: 161-0700 )
N, N, N’, N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
Primer mix (see Recipes)
Cy5-TS primer (see Recipes)
10x TRAP reaction buffer (see Recipes)
50x dNTP (see Recipes)
NP-40 lysis buffer (see Recipes)
Equipment
Polymerase Chain Reaction (PCR) Thermo Cycler (Bio-Rad Laboratories, model: PTC-1148 )
Typhoon PhosphorImager® scanner system (Amersham Biosciences, GE Healthcare, model: Typhoon TRIO )
Software
ImageQuant Software (Molecular Dynamics)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mender, I. and Shay, J. W. (2015). Telomerase Repeated Amplification Protocol (TRAP). Bio-protocol 5(22): e1657. DOI: 10.21769/BioProtoc.1657.
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Category
Cancer Biology > Replicative immortality > Cell biology assays
Cancer Biology > Replicative immortality > Cell biology assays
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1,658 | https://bio-protocol.org/exchange/protocoldetail?id=1658&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Telomere Restriction Fragment (TRF) Analysis
Ilgen Mender
Jerry W. Shay
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1658 Views: 20439
Edited by: HongLok Lung
Reviewed by: Vanesa Olivares-Illana
Original Research Article:
The authors used this protocol in Jan 2015
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The authors used this protocol in:
Jan 2015
Abstract
While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al., 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells.
Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al., 1990; Ouellette et al., 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.
Keywords: Telomere Restriction Fragment Telomeres Telomere length
Materials and Reagents
Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 )
25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 )
DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
Proteinase K (QIAGEN, catalog number: 19131 or 19133 )
Enzymes
HhaI 20,000 units/ml(New England BioLabs, catalog number: R0139L )
HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L )
MspI 20,000 units/ml(New England BioLabs, catalog number: R0106S )
HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L )
RsaI 10,000 units/ml(New England BioLabs, catalog number: R0167L )
AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L )
NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S )
Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S )
Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S )
DEPC-treated water (Life Technologies, catalog number: AM9906 )
Note: Currently, it is “Thermo Fisher Scientific, AmbionTM, catalog number: AM9906”.
Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 )
Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 )
Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 )
Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 )
Boric acid (Sigma-Aldrich, catalog number: B6768 )
UltraPureTM Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500-500 )
GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 )
Radiolabelled TRF Marker (Herbert et al., 2003)
DNA marker (Bionexus, catalog number: BN2050 )
Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 )
Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 )
Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 )
Polyvinylpyrrolidone (Sigma-Aldrich, catalog number: PVP40 )
Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P )
dCTP, [α-32P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 µCi (PerkinElmer, catalog number: NEG513Z500UC )
20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15557-036 )
Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 )
10x Buffer M (Roche Diagnostics, catalog number: 11417983001 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001”.
10x PBS buffer-phosphate buffer saline (see Recipes)
1x TAE buffer-Tris-Acetate-EDTA (see Recipes)
5x TBE buffer-Tris-Borate-EDTA (see Recipes)
Hybridization solution (see Recipes)
100x Denhardt solution (see Recipes)
6x Glycerol and Bromophenol blue Loading Dye (see Recipes)
Equipment
Water bath (Thermo Fisher Scientific, model: Isotemp 205 )
Microcentrifuge (Eppendorf, model: 5424 )
Nanodrop (Thermo Fisher Scientific, Nanodrop Technologies, model: ND-1000 UV/Vis Spectrophotometer )
Gel Tank (Thermo Fisher Scientific, model: OwlTM A2 Large Gel Systems for 20 x 25 cm gel size )
Savant Slab Gel dryer (SibGene, model: SGD4050 )
Hybridizer (Bibby Scientific, Techne, model: Hybrigene )
Cylinder (Bibby Scientific, Techne, model: FHB16/FHB15 )
Thermocycler (LABGENE Scientific, Biometra, model: T1 Thermoblock )
G-BOX (Syngene, model: G-BOX F3 )
Power supply (Whatman Biometra, model: 250 EX )
Microwave oven (GE, model: 1540WW002 )
Screen (Molecular Dynamics, model: Kodak Storage Phosphor Screen )
Typhoon PhosphorImager scanner system (Amersham Biosciences, GE Healthcare, model: Typhoon TRIO )
Software
Image Quant® software (Molecular Dynamics)
Graph Pad Prism 6®
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mender, I. and Shay, J. W. (2015). Telomere Restriction Fragment (TRF) Analysis. Bio-protocol 5(22): e1658. DOI: 10.21769/BioProtoc.1658.
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Category
Cancer Biology > Replicative immortality > Cell biology assays
Cancer Biology > Replicative immortality > Cell biology assays
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1,659 | https://bio-protocol.org/exchange/protocoldetail?id=1659&type=0 | # Bio-Protocol Content
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In vitro and in vivo Limiting Dilution Assay for Colorectal Cancer
LA Lauren Agro
CO Catherine A O’Brien
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1659 Views: 24146
Edited by: HongLok Lung
Reviewed by: Justine Marsolier
Original Research Article:
The authors used this protocol in Jan 2014
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Abstract
The in vitro limiting dilution assay is used to determine the colorectal cancer initiating cell (CC-IC) frequency of a CC-IC enriched suspension culture, grown in growth factor enriched serum free media. The in vivo limiting dilution assay is used to determine the colorectal cancer initiating cell frequency of a primary colorectal cancer sample or an established suspension cell line using immunocompromised murine xenograft models. In vitro and in vivo limiting dilution assays (LDAs) can be used to determine the effect of a specific treatment or genetic knockdown strategy on the initiating cell frequency of a population of CC-ICs or colorectal cancer sample, respectively.
Part I. In vitro CRC LDA
Materials and Reagents
Centrifuge tubes (BD Falcon, catalog numbers: 352096 and 352070 )
Note: Currently, it is “Corning Inc., FalconTM, catalog numbers: 352096 and 352070”.
7x sterile, non-tissue culture 96 well U-bottom plates (Corning Inc., FalconTM, catalog number: 351177 )
FACS tubes with 0.35 μm filter (BD Falcon, catalog number: 352235 )
Note: Currently, it is “Corning Inc., FalconTM, catalog number: 352235”.
Wash media (DMEM/F-12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11320-033 )
0.4% Trypan blue solution (Life Technologies, catalog number: 15250-061 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
Permanent marker (VWR International, catalog number: 52877-310 )
0.25% Trypsin (Life Technologies, catalog number: 25200-056 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
Penicillin-streptomycin-amphotericin B (1%) (Life Technologies, catalog number: 15240-062 )
Note: Currently, it is “Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062)".
L-glutamine (2 mM) (Invitrogen, catalog number: 25030081 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25030081”.
Non-essential amino acids (1x) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023801 )
Sodium pyruvate (1 mM) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023901 )
HEPES (1x) (Invitrogen, catalog number: 15630080 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630080”.
Heparin sodium salt (4 μg/ml) from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3149-100KU )
Lipids (Sigma-Aldrich, catalog number: L0288 )
Human Epidermal Growth Factor Recombinant (EGF) (20 ng/ml) (Biomartcanada, catalog number: RKP01133 )
Human basic Fibroblast Growth Factor Recombinant (bFGF) (10 ng/ml) (Biomartcanada, catalog number: RKP09038 )
N2 supplement-A (STEMCELL Technologies, catalog number: 07152 )
NeuroCult SM1 Neuronal (STEMCELL Technologies, catalog number: 05711 )
Sytox blue (Life Technologies, catalog number: S11348 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S11348”.
Phosphate-buffered saline (PBS) (Life Technologies, catalog number: 10010023 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10010023”.
2% fetal bovine serum (FBS) (Life Technologies, catalog number: 26140-111 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 26140-111 ”.
Serum free cell culture media (see Recipes)
Equipment
37 °C Incubator
Light microscope (Leica Microsystems, model: DM IL LED )
Hemocytometer (Hausser Scientific, catalog number: 1483 )
Pipette aid (INTEGRA Biosciences AG, catalog number: 155 000 )
Pipettes (BD Falcon, catalog number: 357530 )
Note: Currently, it is “Corning Inc., FalconTM, catalog number: 357530”.
Flow cytometry assisted cell sorter (FACS)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cancer Biology > Cancer stem cell > Tumor formation
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166 | https://bio-protocol.org/exchange/protocoldetail?id=166&type=1 | # Bio-Protocol Content
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Subcutaneous Injection of Tumor Cells
JR Jason Reuter
Published: Dec 20, 2011
DOI: 10.21769/BioProtoc.166 Views: 57463
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Abstract
Growth of cells in the subcutaneous space of immunocompromised mice is a common method for assaying tumorigenic potential in vivo. This technique is also used to assess the effects of therapeutic interventions on cancer cell lines.
Materials and Reagents
Tumor cells
CB17 scid/scid mice
Trypsin (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
DMEM (Life Technologies, InvitrogenTM, catalog number: 11965-092 )
Fetal bovine serum (FBS) (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
Phosphate buffered saline (PBS)
Trypan blue (Life Technologies, InvitrogenTM, catalog number: 15250-061 )
Isoflurane (usually purchased through animal facility at institution)
Matrigel (BD Biosciences, catalog number: 356234 )
DMEM cultural medium (see Recipes)
Equipment
Centrifuges
Insulin syringe
Hemocytometer
Cell culture hood
Incubator
Microscope
Small gauge
Eppendorf tube
Procedure
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Reuter, J. (2011). Subcutaneous Injection of Tumor Cells. Bio-101: e166. DOI: 10.21769/BioProtoc.166.
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Category
Cancer Biology > General technique > Animal models > Cell invasion
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1,660 | https://bio-protocol.org/exchange/protocoldetail?id=1660&type=0 | # Bio-Protocol Content
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Purification of Bacterial RNA from Infected Macrophages
LL Lior Lobel
NS Nadejda Sigal
AP Anna Pasechnek
AH Anat A. Herskovits
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1660 Views: 10061
Edited by: Fanglian He
Reviewed by: Yoko Eguchi
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or microarray. We have successfully employed this protocol in our studies of Listeria monocytogenes gene regulation during infection in vitro (Lobel et al., 2015; Lobel et al., 2012; Kaplan Zeevi et al., 2013; Rabinovich et al., 2012).
Keywords: Listeria monocytogenes Bacterial RNA extraction Intracellular bacterial RNA
Materials and Reagents
Cell scrapers (Thermo Fisher Scientific, NuncTM, catalog number: 179693 )
MF-Millipore filters (Merck Millipore Corporation, catalog number: HAWP04700 )
Cell culture dishes (Greiner Bio One International GmbH, catalog number: 639160 )
Eppendorf tubes (Corning Inc., Axygen, catalog number: MCT175C )
Pipettes sterile (25 ml) (Greiner Bio One International GmbH)
Falcon tubes (50 ml) (Greiner Bio One International GmbH)
Listeria monocytogenes 10403S (Daniel Portnoy’s lab stock) (Becavin et al., 2014)
Bone marrow (from C57B/6 female mice; ordered from Harlan labs Israel)
Liquid nitrogen
RNase-free water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 41965039 )
L-Glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360088 )
2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Gentamicin (Sigma-Aldrich, catalog number: G1397 )
Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8537 )
Brain Heart Infusion (BHI) (Merck Millipore Corporation, catalog number: 1104930500 )
Phenol saturated (pH 4.3) (Thermo Fisher Scientific, catalog number: BP1751I-400 )
Chloroform (Thermo Fisher Scientific, catalog number: BP1145-1 )
Isoamyl alcohol (Sigma-Aldrich, catalog number: W205702 )
Sodium acetate Anhydrous (Sigma-Aldrich, catalog number: W302406 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
DNase I, RNase-free (supplied with MnCl2) (1 U/µl) (Fermentas, catalog number: EN0521 )
Note: Currently, it is “Thermo Fisher Scientific, catalog number: EN0521”.
10% Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4522 )
Ethanol absolute (Merck Millipore Corporation, catalog number: 1070174000 )
M-CSF (L-929 conditioned medium) (Englen et al., 1995)
Note: Alternatively M-CSF can be bought from Sigma (Sigma-Aldrich, catalog number: SRP3221 ).
BMDM + PS media (filter sterilized) (see Recipes)
BMDM no PS media (filter sterilized) (see Recipes)
AE buffer (see Recipes)
Phenol-chloroform-IAA (see Recipes)
Chloroform-IAA (see Recipes)
Equipment
CO2 forced-air incubator (Thermo Fisher Scientific, model: 3111 )
Kontes glass holder (Thermo Fisher Scientific, catalog number: K953755-0045 )
Speed-Vac Concentrator (Thermo Fisher Scientific, catalog number: SPD131DDA )
Vortex-Genie 2 (Scientific Industries, model: G560E )
Nanodrop 1000 (Thermo Fisher Scientific)
30 °C incubator (Thermo Fisher Scientific)
65 °C heat block (Thermo Fisher Scientific)
4 °C table centrifuge (Thermo Fisher Scientific, EppendorfTM, model: 5417R )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lobel, L., Sigal, N., Pasechnek, A. and Herskovits, A. A. (2015). Purification of Bacterial RNA from Infected Macrophages. Bio-protocol 5(22): e1660. DOI: 10.21769/BioProtoc.1660.
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Category
Microbiology > Microbial genetics > RNA
Microbiology > Microbe-host interactions > In vitro model
Molecular Biology > RNA > RNA extraction
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1,661 | https://bio-protocol.org/exchange/protocoldetail?id=1661&type=0 | # Bio-Protocol Content
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Skin Wound Healing Model - Excisional Wounding and Assessment of Lesion Area
Camila Francisco Moreira
Puebla Cassini-Vieira
MS Mariana Felipetto da Silva
Lucíola da Silva Barcelos
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1661 Views: 23200
Edited by: Ivan Zanoni
Reviewed by: Achille BroggiMarco Di Gioia
Original Research Article:
The authors used this protocol in Nov 2014
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Abstract
This protocol focus on the most common surgical mouse model of cutaneous excisional wound healing used to study the cellular and molecular pathways involved in wound repair and regeneration as well as in translational applications such as the evaluation of new therapeutic modalities. This model allows the monitoring of the wound closure and the tissue collection for histological and molecular analyses. Briefly, full skin thickness excisional wounds are created on the dorsum of the mouse as the excision extends through the panniculosus carnosus. Wounds larger and minor diameters are then regularly measured and wound closure rate is calculated based on wound area relative to the original size.
Materials and Reagents
5 mm diameter circular biopsy punch (ABC, catalog number: 0418 )
10% Ketamine Hydrochloride (Agropecuária Tarumã, catalog number: 8565 )
2% Xylazine hydrochloride (Syntec)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Saline solution (0.9% sodium chloride injectable solutio) (Equiplex)
dH2O
70% alcohol (see Recipes)
Phosphate buffered saline (PBS) (pH 7.2, 10x) (see Recipes)
Phosphate buffered saline (PBS) (pH 7.2, 1x) (see Recipes)
Equipment
Digital caliper (Mitutoyo, catalog number: 573-661 )
Hair removal machine (Wahl and Toshico)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Moreira, C. F., Cassini-Vieira, P., da Silva, M. F. and Barcelos, L. D. S. (2015). Skin Wound Healing Model - Excisional Wounding and Assessment of Lesion Area. Bio-protocol 5(22): e1661. DOI: 10.21769/BioProtoc.1661.
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Category
Immunology > Animal model > Mouse
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1,662 | https://bio-protocol.org/exchange/protocoldetail?id=1662&type=0 | # Bio-Protocol Content
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Estimation of Wound Tissue Neutrophil and Macrophage Accumulation by Measuring Myeloperoxidase (MPO) and N-Acetyl-β-D-glucosaminidase (NAG) Activities
Puebla Cassini-Vieira
Camila Francisco Moreira
MS Mariana Felipetto da Silva
Lucíola da Silva Barcelos
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1662 Views: 13398
Edited by: Ivan Zanoni
Reviewed by: Achille BroggiMarco Di Gioia
Original Research Article:
The authors used this protocol in Nov 2014
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Abstract
The inflammatory response is essential to the reestablishment of cutaneous homeostasis following injury. In this context, leukocytes arrive at the wound site and orchestrate essential events in the wound healing process. Therefore, the quantification of specific subsets of inflammatory cells in the wound tissue is of considerable interest. The current protocol focus on a quantitative index of neutrophils and macrophages accumulation within skin lesions by measuring the specific activity of the marker enzymes Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG), respectively. MPO is present in high levels in the azurophilic granules of neutrophils and NAG in lysosomes of activated macrophages. These methods allow the indirect estimation of the abundance of neutrophils and macrophages accumulated into the skin.
Materials and Reagents
2 ml microcentrifuge tubes (Eppendorf)
Circular biopsy punch (ABC, catalog number: 0418 )
96-well microplates (Thermo Fisher Scientific, FisherbrandTM, catalog number: 21-377-203 )
3% Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: H1009 )
3, 3’-5, 5’-tetramethylbenzidine (TMB) (Sigma-Aldrich, catalog number: T2885 )
4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, catalog number: N9376 )
Citric acid (Sigma-Aldrich, catalog number: 251275 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
Glucose Solution (Sigma-Aldrich, catalog number: G8644 )
Sulfuric acid (H2SO4) (Sigma-Aldrich, catalog number: 258105 )
Hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich, catalog number: H9151 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
Liquid nitrogen
Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA) (Sigma-Aldrich, catalog number: V000114 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
Sodium phosphate tribasic dodecahydrate (Na3PO4) (Sigma-Aldrich, catalog number: 222003 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Phosphate buffered saline (PBS) (10x, pH 7.2) (see Recipes)
PBS (1x) (see Recipes)
Buffer 1 (see Recipes)
Buffer 2 (see Recipes)
H2O2 (0.002% v/v) (Sigma-Aldrich, catalog number: 7722-84-1 ) (see Recipes)
MPO substrate solution (see Recipes)
Saline-Triton buffer (see Recipes)
Phosphate-citrate buffer (see Recipes)
NAG substrate solution (see Recipes)
Glycine buffer (see Recipes)
Equipment
Freezer (-20 °C)
Freezer (-80 °C)
High Precision Laboratory Balance (Shimadzu Corporation, catalog number: ATX -224 )
Micropipettes
Microplate Spectrophotometer Reader (Molecular Devices, model: SpectraMax )
Refrigerated centrifuge (Eppendorf, model: 5810R )
Tissue homogenizer (Thermo Fisher Scientific, catalog number: PowerGen 125 )
Vortex Mixer (VELP Scientifica, catalog number: F202A0171 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Immune cell function > Macrophage
Immunology > Immune cell function > Neutrophil
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1,663 | https://bio-protocol.org/exchange/protocoldetail?id=1663&type=0 | # Bio-Protocol Content
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Permanent Occlusion of the Left Anterior Coronary Artery in the Rat
MB Mélanie Bousquenaud
PM Pierre-Yves Marie
Yvan Devaux
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1663 Views: 8400
Edited by: Ivan Zanoni
Reviewed by: Martin V Kolev
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
Left ventricular (LV) remodeling occurs in many patients after myocardial infarction (MI). LV remodeling is characterized by progressive ventricular dilatation and contractile dysfunction, consequently to cardiomyocyte hypertrophy and fibrosis. Despite reperfusion therapies, this pathophysiological process is the main cause of cardiac evolution toward heart failure. Moreover, the outcome of patients after MI is largely dependent on the initial cardiac injury. Thus, this is of major clinical interest to develop new pharmacological strategies to limit infarct size and prevent or reverse left ventricular remodeling. Such preclinical cardiovascular treatments are often tested in rodents. The rat model of myocardial infarction is commonly used. In this model, the permanent ligation of the left anterior descending coronary artery is performed (Bousquenaud et al., 2013a).
After being used to this surgical technique and experimented, the operator will need 20 min per rat from the anesthesia to the rat recovering.
Materials and Reagents
Sterile drapes
Non-resorbable silk: Prolene 7.0 (Ethicon, catalog number: F1839 )
Resorbable silk: Vicryl 4.0 (Ethicon, catalog number: V134 )
Syringes (BD Biosciences, catalog number: 309659 )
2 shoelaces (around 20 cm)
Tracheal tube (Fine Science Tools, catalog number: RSP-ETT1605 )
Endotracheal tube (Kent Scientific Corporation, catalog number: RSP-ETT1605 )
Rat Adult male Wistar rats weighing around 300 g (Charles River Laboratories International)
Dermic Betadine 10%
Antibiotics: Amoxicilline (Clamoxyl 100 mg/kg/24 h)
Trichrome stain (Masson) Kit (Sigma-Aldrich, catalog number: HT15 )
Anti-sarcomeric alpha actinin antibody [EA-53] (Abcam, catalog number: ab9465 )
Equipment
Gaseous anesthesia delivery system (Minerve)
Ventilator (Kent Scientific Corporation, model: TOPO220 )
Fine curved forceps (Fine Science Tools, catalog number: 11272-30 )
Shaver (Kent Scientific Corporation, catalog number: CL8787 )
Rasor blade (Fine Science Tools, catalog number: 10008-13 )
Scalpel (Fine Science Tools, catalog number: 10011-00 )
Needle holders (Fine Science Tools, catalog number: 12001-13 )
Retractor (Fine Science Tools, catalog number: 17012-11 )
Procedure
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Category
Immunology > Animal model > Rat
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1,664 | https://bio-protocol.org/exchange/protocoldetail?id=1664&type=0 | # Bio-Protocol Content
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Peer-reviewed
Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS
Nina Chumak
MM Magdalena Mosiolek
Vera Karolina Schoft
Published: Vol 5, Iss 22, Nov 20, 2015
DOI: 10.21769/BioProtoc.1664 Views: 12587
Edited by: Samik Bhattacharya
Reviewed by: Yurong XiePengpeng Li
Original Research Article:
The authors used this protocol in Mar 2015
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Mar 2015
Abstract
One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 106 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.
Keywords: SYBR Green Fluorescence-activated cell sorting (FACS) Arabidopsis Pollen Male gametophyte
Materials and Reagents
Plastic ware / consumables
15 ml polypropylene tubes (SARSTEDT AG & Co, catalog number: 62554502 )
50 ml polypropylene tubes (SARSTEDT AG & Co, catalog number: 62548004 )
100 µm nylon mesh (Biologix Group Limited, catalog number: 151100 )
40 µm nylon mesh (Biologix Group Limited, catalog number: 151040 )
250 ml centrifuge tubes (Bartelt, catalog number: 9.315 721 )
1.5 ml and 2 ml microliter reaction tubes
Syringe filters 0.2 µm (Bartelt, catalog number: 9055511 )
BD Falcon® 12 x 75 mm tube with cell strainer cap (BD Biosciences, catalog number: 352235 )
Note: Currently, it is “Corning Inc., catalog number: 352235”.
Sterican disposable hypodermic needles with a diameter of 0.45 x 25 mm (B. Braun Melsungen AG, Braun Petzold, catalog number: 4657683 )
Pipettes tips (1,000 µl, 200 µl, 20 µl, 2 µl)
Acid-washed glass beads (see Recipes)
Glassware
Glass beads 0.4-0.6 mm (Sartorius AG, catalog number: BBI8541701 )
250 ml Erlenmeyer flask
Glass tubes
Glass slides
Cover slips
Plant material
100 Flowering wild-type Arabidopsis thaliana plants (Col-0 accession and dml-3 mutant, are used as examples) (Arabidopsis, catalog number: SALK_056440 )
Chemicals
D(+)-Sucrose for analysis (AppliChem GmbH, catalog number: 131621.0914 )
DAPI (4', 6-Diamidino-2-Phenylindole, dihydrochloride) (Life Technologies, InvitrogenTM, catalog number: D1306 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306”.
SYBR Green I nucleic acid stain stock solution (Life Technologies, catalog number: S7567 )
Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: S7567”.
Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: 93482 )
Complete, EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11873580001 )
Calcium chloride dihydrate (AppliChem GmbH, catalog number: A3587 )
Ethylenediamine tetraacetic acid disodium salt dihydrate (Carl Roth GmbH + Co., catalog number: 8043 )
Ethylene glycol-bis (2-aminoethylether)-N, N, N, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
Ficoll® PM 400 (Sigma-Aldrich, catalog number: F4375 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Magnesium acetate tetrahydrate (Sigma-Aldrich, catalog number: M5661 )
Sodium chloride (EMD Millipore Corporation, catalog number: 106404 )
Potassium chloride (Sigma-Aldrich, catalog number: P3911 )
D-Sorbitol (Sigma-Aldrich, catalog number: S6021 )
Spermine [N, N'-Bis-(3-aminopropyl)-1, 4-diaminobutan] (AppliChem GmbH, catalog number: A0672.0005 )
TritonTM X-100 (Sigma-Aldrich, catalog number: 234729 )
Tris-base (AppliChem GmbH, catalog number: A1086 )
Nitric acid 65% (Carl Roth GmbH + Co., catalog number: X898.1 )
Hydrochloric acid fuming 37% (Carl Roth GmbH + Co., catalog number: 4625.1 )
Sodium Hydroxide pellets (AppliChem GmbH, catalog number: A3910 )
Ultrapure water (MilliQ) (conductivity > 18 MΩ-1 cm-1)
Buffers
BD FACSFlow Sheath Fluid (BD Bioscience, catalog number: 342003 )
Sucrose solution (see Recipes)
Buffer A (see Recipes)
Buffer B (see Recipes)
Protease inhibitor stock solution (see Recipes)
DAPI stock solution (see Recipes)
Equipment
Instruments
Beckman Coulter Avanti J-26XP centrifuge with the JA-10 rotor
Table-top centrifuge
Orbital shaker (VWR International, catalog number: 89032102 )
Drying oven (EHRET GmbH, catalog number: 3108 )
Bead-beater (Retsch GmbH, model: MM301 )
Fluorescence microscope with 20x, 40x, and 100x objectives and a DAPI filter
Flow cytometer: FACS Aria I (BD Biosciences) with a 70 µm nozzle and 70 psi sheath pressure, and a 488 nm coherent sapphire solid state 13 mW laser for excitation and a 530/30 nm band-pass filter for detection of SYBR green I
General lab equipment
Beaker
250 ml Erlenmeyer flask
Glass tubes
Sharp-edged scissors
Forceps
Air displacement pipettes
Ice container
Software
Flow cytometric software (BD Biosciences, model: FACS Diva 6.1.2)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant cell biology > Organelle isolation
Cell Biology > Cell-based analysis > Flow cytometry
Cell Biology > Cell staining > Nucleic acid
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1,665 | https://bio-protocol.org/exchange/protocoldetail?id=1665&type=0 | # Bio-Protocol Content
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Peer-reviewed
Quantification of the Volume and Surface Area of Symbiosomes and Vacuoles of Infected Cells in Root Nodules of Medicago truncatula
Aleksandr Gavrin
EF Elena E. Fedorova
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1665 Views: 10376
Edited by: Arsalan Daudi
Reviewed by: Renate Weizbauer
Original Research Article:
The authors used this protocol in Sep 2014
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Sep 2014
Abstract
Legumes are able to form endosymbiotic interactions with nitrogen-fixing rhizobia. Endosymbiosis takes shape in formation of a symbiotic organ, the root nodule. Medicago truncatula (M. truncatula) nodules contain several zones representing subsequent stages of development. The apical part of the nodule consists of the meristem and the infection zone. At this site, bacteria are released into the host cell from infection threads. Upon release, bacteria are surrounded by a host cell-derived membrane to form symbiosomes. After release, rhizobia grow, divide, and gradually colonize the entire host cell of the fixation zone of root nodules. Therefore, mature infected cells contain thousands of symbiosomes, which remain as individual units among other organelles. Visualization of the organization and dynamics of the symbiosomes as well as other organelles in infected cells of nodules is essential to understand mechanisms regulating the development of endosymbiosis between plants and rhizobia. To examine this highly dynamic developmental process, we designed a useful imaging technique that is based on confocal scanning microscopy combined with different fluorescent dyes and GFP-tagged proteins (Gavrin et al., 2014). Here, we describe a protocol for microscopic observation, 3D rendering, and volume/area measurements of symbiosomes and other organelles in infected cells of M. truncatula root nodules. This protocol can be applied for monitoring the development of different host-microbe interactions whether symbiotic or pathogenic.
Keywords: Symbiosis Cell imaging 3D rendering Organelle visualization Cell measurements
Materials and Reagents
Microscope cover glasses
Microscope slides
Perlite (Maasmond-Westland, The Netherlands)
7 days old seedlings of M. truncatula (seeds can be obtained at The Samuel Roberts Noble Foundation)
Agrobacterium rhizogenes MSU440 strains (can be obtained at the Laboratory of Molecular Biology, Wageningen University, The Netherlands) containing pUB:GFP-MtVTI11 or pLB:GFP-MtVTI11 binary vector (see Note 1)
Sinorhizobium meliloti 2011 (see Note 2)
0.1 M sodium phosphate (Sigma-Aldrich, catalog numbers: S8282 and S7907 ) buffer (pH 7.2) with 3% sucrose (Sigma-Aldrich, catalog number: S7903). For preparation protocol see Sambrook and Russell (2001)
Note: Sodium phosphate monobasic (Sigma-Aldrich, catalog number: S8282) and Sodium phosphate dibasic (Sigma-Aldrich, catalog number: S7907)
10 μg/ml Propidium iodide (store at 4 °C) (Sigma-Aldrich, catalog number: P4170 )
1% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15700 )
0.75% glutaraldehyde (Electron Microscopy Sciences, catalog number: 16000 )
50 mM phosphate buffer (Sambrook and Russell, 2001)
Fixative (see Recipes)
Equipment
Fluorescence stereomicroscope (Leica Microsystems, model: MZFLIII )
Confocal laser scanning microscope with digital camera (ZEISS, model: Axiovert 100M )
Razor blades
Fine tweezers (Structure Probe, Dumont, model: 0T05B-XD )
Software
IMARIS software (Bitplane) with modules for data management, visualization, 3D rendering and analysis
Zeiss LSM Image Browser
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant physiology > Endosymbiosis
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Confocal microscopy
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1,666 | https://bio-protocol.org/exchange/protocoldetail?id=1666&type=0 | # Bio-Protocol Content
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Peer-reviewed
Single Molecule RNA FISH in the Mammalian Oocyte
Denisa Jansova
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1666 Views: 10244
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
RNA fluorescence in situ hybridization is a method to localize and measure gene expression in individual cell or tissue. Using multiple specific fluorescently labeled oligonucleotides greatly increases signal-to-noise ratio and thus enables detection of single RNA molecule. Around forty different DNA oligonucleotides designed to common RNA target and labeled with single fluorophore at 3´ terminus hybridizes with target RNA in fixed cells. We adapt this method to visualize target RNA in the mammalian oocyte. The ability to detect single transcript in the mammalian oocyte was challenging due to its large cell size. This method consists of four simple steps: fixation, permeabilization, hybridization and imaging. The protocol is adapted to this large nonattached cell to visualize maternal RNAs.
Combination of various fluorophores allows detection of more RNA targets. This method might be used with organelle markers or expanded with immunofluorescence protocol.
Keywords: Oocyte Visualization RNA Fluorescent in situ hybridization Maternal RNA Gene expression
Materials and Reagents
Cover glasses thickness No.1 22 x 22 mm (Marienfeld-Superior, catalog number: 0101050 )
Fisherfinest Premium Microscope Slides (Thermo Fisher Scientific, catalog number: 125447 )
Tissue culture 24-well plates (TPP Techno Plastic Products AG, catalog number: 92024 )
Tissue culture 96-well plates (TPP Techno Plastic Products AG, catalog number: 92096 )
Borosilicate glass capillary 3.3. (Hilgenberg GmbH, catalog number: 1409363 ), hand pipette for manipulation) with tip diameter around 100 µm (Hilgenberg GmbH)
Microtube from Eppendorf 15 ml RNase free (Sigma-Aldrich, catalog number: 0030123.328 )
Note: Pricing & availability is not currently available.
Aluminum foil (29 cm x 150 m) (MAIS.s.r.o.)
Oocytes from Six-weeks-old female CD1 mice; isolation of mouse oocytes described in Bio-protocol (Tetkova and Hancova, 2015), also (Susor et al., 2015)
Paraformaldehyde final concentration 4% in PBS (store at -20 °C) (Thermo Fisher Scientific, Afla Aesar, catalog number: 30525894 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 30525894 ”.
Triton X-100 final concentration 2% in 1x PBS (fresh) (Sigma-Aldrich, catalog number: 9002931 )
20x saline-sodium citrate (SSC) stock is diluted in RNase free water, in the same day of experiment (Sigma-Aldrich, catalog number: S6639 )
Ethanol 100% (for final concentration 70% ethanol, freshly dilute 100% ethanol with DEPC water) (Merck Millipore Corporation, catalog number: 1085430250 )
VECTASHIELD HardSetTM with DAPI (storage 2-8 °C) (Vector Laboratories, catalog number: H1500 )
ProtectRNA RNase Inhibitor 500x concentrated (final working concentration is 1x; storage at 2-8 °C in dark) (Sigma-Aldrich, catalog number: R7397 )
Stellaris probes (stock concentration 5 nmol) (Bioresearch Technologies)
Nuclease-free water (Life Technologies, Ambion®, catalog number: AM9932 )
Note: Currently, it is “Thermo Fisher Scientific, AmbionTM, catalog number: AM9932”.
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417 )
Poly(vinyl alcohol) (PVA) (Sigma-Aldrich, catalog number: 341584 )
Formamide (5 ml for 10% final concentration) (Sigma-Aldrich, catalog number: F7503 )
Dextran sulfate sodium salt from Leuconostoc spp. (Sigma-Aldrich, catalog number: D8906 )
Wash buffer (see Recipes)
Hybridization buffer (see Recipes)
Isolation buffer PVA (see Recipes)
Equipment
Stereomicroscope Zeiss 2000C (Thermo Fisher Scientific)
Heating plate (P-lab)
Multi Bio 3D mini-Shaker (Biosan Laboratories)
Thermostat incubator mini I5110 (Labnet International)
Confocal microscope Leica SP5 (Leica Microsystems)
Filter sets appropriate for fluorophores and excitation lasers
40x or 63x or 100x oil immersion objective
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Jansova, D. (2015). Single Molecule RNA FISH in the Mammalian Oocyte. Bio-protocol 5(23): e1666. DOI: 10.21769/BioProtoc.1666.
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Category
Cell Biology > Cell staining > Nucleic acid
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1,667 | https://bio-protocol.org/exchange/protocoldetail?id=1667&type=0 | # Bio-Protocol Content
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Peer-reviewed
Establishing a Biofilm Co-culture of Pseudomonas and Aspergillus for Metabolite Extraction
HZ He Zheng
NK Nancy P. Keller
YW Yun Wang
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1667 Views: 11079
Edited by: Arsalan Daudi
Reviewed by: Timo Lehti
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
Filamentous fungi and bacteria form mixed-species biofilms in nature and diverse clinical contexts (Frey-Klett et al., 2011; Peleg et al., 2010). The interactions between fungi and bacteria, often mediated by secreted metabolites, have important ramifications for the biology of the interacting partners (Frey-Klett et al., 2011). This is particularly true for the bacterium Pseudomonas aeruginosa (P. aeruginosa) and the fungus Aspergillus fumigatus (A. fumigatus) which often reside in the same niche such as lungs of cystic fibrosis (CF) patients. Some studies have reported that co-infection with P. aeruginosa and A. fumigatus could lead to a decrease in lung function relative to their respective single species infection (Amin et al., 2010; Peleg et al., 2010). Metabolite extraction and analysis allow for the characterization of specific microbial metabolites in the polymicrobial biofilm. This protocol describes how to prepare the Pseudomonas-Aspergillus co-culture biofilm on solid medium in preparation for metabolite extraction.
Keywords: Aspergillus fumigatus Pseudomonas aeruginosa Biofilm Cross Kingdom communication
Materials and Reagents
Plastic spreader (Excel Scientific, catalog number: 5PR-L-510 )
Falcon tubes (15 ml) (Corning Inc., catalog number: 352196 )
Petri dish (100 x 15 mm) (Corning Inc., catalog number: 351029 )
Petri dish (60 x 15 mm) (Corning Inc., catalog number: 351007 )
Inoculating needle (VWR International, catalog number: 12000-816 )
Glass vial (10 ml) (VWR International, catalog number: SC66022-300 )
Aluminum foil
Pseudomonas aeruginosa culture
Aspergillus fumigatus culture
D-glucose (Thermo Fisher Scientific, Alfa Aesar, catalog number: A1682836 )
Anhydrous dextrose (VWR International, BDH, catalog number: BDH9230500G )
Yeast extract (BD, BactoTM, catalog number: 288620 )
Agar-agar (Merck Millipore Corporation, catalog number: EM1.01614.0500 )
Peptone (BD, BactoTM, catalog number: 212120 )
Luria-Bertani (LB) dehydrated medium (Merck Millipore Corporation, catalog number: 1.10285.5000 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
GR ACS Sodium Nitrate (NaNO3) (Merck Millipore Corporation, catalog number: SX06551 )
NaCl
Potassium Chloride (KCl) (VWR International, BDH, catalog number: 0395VBD500G )
Potassium dihydrogen phosphate (KH2PO4) (Thermo Fisher Scientific, Alfa Aesar, catalog number: 1159436 )
Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: M1880 )
Copper(II) sulfate pentahydrate (CuSO4.5H2O) (Sigma-Aldrich, catalog number: C8027 )
Zinc sulfate heptahydrate (ZnSO4.7H2O) (Sigma-Aldrich, catalog number: Z0251 )
Ammonium molybdate tetrahydrate [(NH4)6Mo7O24.4H2O] (Sigma-Aldrich, catalog number: 431346 )
Manganese(II) sulfate monohydrate (MnSO4.H2O) (Sigma-Aldrich, catalog number: M7634 )
Iron(II) sulfate heptahydrate (FeSO4.7H2O) (Sigma-Aldrich, catalog number: F8633 )
Ethylenediaminetetraacetic acid disodium salt dehydrate (Na2EDTA.2H2O) (Sigma-Aldrich, catalog number: E4884 )
Cobalt(II) chloride hexahydrate (CoCl2.6H2O) (Sigma-Aldrich, catalog number: 255599 )
Tween 20 solution (Sigma-Aldrich, catalog number: P2287 )
Chloroform (VWR International, BDH, catalog number: PL049ZA4 )
Acetonitrile (Honeywell International Inc., catalog number: AH015-4 )
PBS-0.01 % Tween 20 buffer (see Recipes)
20x sodium nitrate salts solution (see Recipes)
Trace elements (see Recipes)
Medium recipes (see Recipes)
Solid Luria-Bertani (LB) dehydrated medium (see Recipes)
Solid yeast extract-peptone-dextrose medium (YPD) (see Recipes)
Equipment
Incubator (VWR International, model: 1535 )
Orbital shaker (Eppendorf AG, New Brunswick Scientific, model: I26 )
Centrifuge (Beckman, model: J20 ) with rotor (Beckman, model: JLA-10.500 )
Microscope (Celestron, model: 44345 )
Hemocytometer (Hausser Scientific, catalog number: 3500 )
Steel corer (38.1 mm in diameter) (Figure 1)
Figure 1. Steel corer
Steel blade (VWR International, catalog number: 55411-050 )
Steel pestle (VWR International, catalog number: 62400-336 )
0.2 μm-pore-size hydrophilic filter (VWR International, catalog number: 28145-477 )
Glass culture tube (15 ml) (VWR International, catalog number: 47729-583 )
Glass centrifuge tube (15 ml) (SP Industries, Wilmad-LabGlass, catalog number: C-8060-15 )
Nitrogen gas cylinder
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zheng, H., Keller, N. P. and Wang, Y. (2015). Establishing a Biofilm Co-culture of Pseudomonas and Aspergillus for Metabolite Extraction. Bio-protocol 5(23): e1667. DOI: 10.21769/BioProtoc.1667.
Download Citation in RIS Format
Category
Microbiology > Microbial biofilm > Biofilm culture
Microbiology > Microbial cell biology > Cell isolation and culture
Microbiology > Microbial metabolism > Other compound
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1,668 | https://bio-protocol.org/exchange/protocoldetail?id=1668&type=0 | # Bio-Protocol Content
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Peer-reviewed
Excision of Visceral Adipose Tissue from Live Mice (VATectomy)
Marko Šestan
FW Felix M. Wensveen
Bojan Polić
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1668 Views: 13355
Reviewed by: Shanie Saghafian-Hedengren
Original Research Article:
The authors used this protocol in Apr 2015
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Abstract
The visceral adipose tissue (VAT) has been shown to play an important role in various biological functions. It is a storage depot for nutrients and it is an important endocrine organ producing hormones that control systemic metabolism (McGown et al., 2014). Importantly, following diet-induced obesity, VAT accumulates a large number of activated immune cells, which produce cytokines that drive chronic systemic inflammation and promote insulin resistance (Johnson et al., 2012; Wensveen et al., 2015). VAT therefore plays a key role in metabolic, endocrinological and immunological research. To show the importance of this organ in various research models, one may surgically remove the organ in a procedure called VATectomy. This protocol describes the technical procedures required for an efficient VATectomy of the perigonadal fat pads in mice.
Keywords: Visceral Adipose Tissue Diabetes Obesity Inflammation Excision
Materials and Reagents
Male Mouse (e.g., C57BL/6, typically of 6-12 weeks old) (male mice are more severely affected by type 2 diabetes than female mice, and they are used exclusively in diet-induced diabetes studies) (Singer et al., 2015).
Skin disinfectant [e.g., Chlorhexidine gluconate (generic name: Pliva®sept pjenušavi) (PLIVA HRVATSKA, catalog number: 536-02884 )
Non-Steroid anti-inflammatory drugs (NSAIDs) [e.g., Meloxicam (generic name: loxicum) (5 mg/ml) (Norbrook Laboratories, catalog number: SC-200626 )]
Inhalation anesthetics [e.g., Forane (generic name: isofluranum) (Abbott Laboratories, catalog number: B506 ).
Sterile physiological salt solution (0.9% NaCl)
Depilation cream (Depilation) (Afrodita kozmetika, catalog number: 05-506 )
Meloxicam working solution (see Recipes)
Equipment
Inhalation anesthesia equipment (alternatively, one may use intravenous anesthesia)
Basic surgical instruments (e.g., needle holder, tweezers, scissors, scalpel, hemostat, retractor, surgical needle)
(Absorbable) suture materials [e.g., Coated VicrylTM, size 3-0 (Ethicon, model: JB944 ) or stapler [e.g., Reflex, 9 mm applier (AgnTho's, model: 202-1000 )]
Hair trimmer
Scale
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Šestan, M., Wensveen, F. M. and Polić, B. (2015). Excision of Visceral Adipose Tissue from Live Mice (VATectomy). Bio-protocol 5(23): e1668. DOI: 10.21769/BioProtoc.1668.
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Category
Immunology > Animal model > Mouse
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1,669 | https://bio-protocol.org/exchange/protocoldetail?id=1669&type=0 | # Bio-Protocol Content
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Isolation of Lymphocytes from Murine Visceral Adipose Tissue
Sonja Valentić
FW Felix M. Wensveen
Bojan Polić
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1669 Views: 16951
Reviewed by: Shanie Saghafian-Hedengren
Original Research Article:
The authors used this protocol in Apr 2015
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Abstract
Several studies have shown that the detrimental influence of abdominal obesity on metabolic processes is mediated by the intra-abdominal fat depot. Visceral adipose tissue has been shown to be an independent risk factor for coronary heart disease, hypertension, impaired glucose tolerance and Diabetes Mellitus Type 2 (DM2). Diet-induced obesity in mice, primarily of the C57BL/6J strain, is a commonly used method to study the development of insulin resistance as a model for DM2. The white or visceral adipose tissue (here referred to as VAT), especially the fat around the gonads, is a commonly used organ of study in this model, as it accumulates large numbers of lymphocytes in response to diet-induced obesity. The protocol below describes the isolation of lymphocytes from the stromal vascular fraction (SVF) from VAT.
Materials and Reagents
50 ml centrifuge tubes
70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
Male mouse (e.g., C57BL/6J) 8-20 weeks old
Note: Generally, male mice are more severely affected by type 2 diabetes than female mice, and they are used exclusively in diet-induced diabetes studies (www.jax.org).
Collagenase from Clostridium histolyticum type IV (Sigma-Aldrich)
Fetal Bovine Serum (FBS) (Pan biotech GmbH)
Trypan blue
RPMI 1640 (with L-glutamine; 25 mM Hepes; 2.2 g/L NaHCO3) (Pan Biotech GmbH)
MilliQ water
0.83% NH4Cl
0.168% Na2CO3
1 mM EDTA (pH 7.3)
1x PBS (pH 7.3)
0.2% BSA
3% RPMI 1640 (see Recipes)
3% RPMI + 1 mg/ml Collagenase D (or IV) (see Recipes)
Erylysis buffer (see Recipes)
FACS wash buffer (see Recipes)
Equipment
Soft wood tablet and pins
Disinfectant
Small thin surgical scissors and tweezers
Thermostatic shaker
Vortex
Vacuum pump
Centrifuge
Procedure
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How to cite：Valentić, S., Wensveen, F. M. and Polić, B. (2015). Isolation of Lymphocytes from Murine Visceral Adipose Tissue. Bio-protocol 5(23): e1669. DOI: 10.21769/BioProtoc.1669.
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Category
Immunology > Immune cell isolation > Lymphocyte
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167 | https://bio-protocol.org/exchange/protocoldetail?id=167&type=0 | # Bio-Protocol Content
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Process and Analysis of Kidney Infiltrates by Flow Cytometry from Murine Lupus Nephritis
Ramalingam Bethunaickan
AD Anne Davidson
Published: Vol 2, Iss 9, May 5, 2012
DOI: 10.21769/BioProtoc.167 Views: 17155
Original Research Article:
The authors used this protocol in Apr 2011
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Abstract
Methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE are essential to understand the patho-physiology of the disease. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans.An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that lead to renal impairment in SLE and other renal inflammatory diseases.
Keywords: SLE nephritis Kidney infiltrates Macrophages from kidney Collagen digestion Inflammation
Materials and Reagents
Fetal bovine serum (FBS)
Sterile PBS (Life Technologies, Invitrogen™, catalog number: 20012-027 )
0.17M Ammonium chloride
Collagenase Type I (CLS I) (Worthington, catalog number: 4197 , specific activity 230 U mg-1)
DMEM, High glucose (Life Technologies, Gibco®, catalog number: 10313 )
Paraformaldehyde (Tousimis, catalog number: 1108A )
BSA (Fraction V) (Sigma-Aldrich, catalog number: A7030 )
Fc block (CD16/CD32)
FACS staining buffer (see Recipes)
Equipment
BD LSRII or similar flow cytometer
Bench-top refrigerated centrifuge
BD cell strainer (40 nm) (BD Biosciences, Falcon®, catalog number: 352340 )
30 ml syringe (BD Biosciences, Falcon®, catalog number: 309661 )
Microscopes
21G Needles (BD Biosciences, Falcon®, catalog number: 305165 )
26G needles (BD Biosciences, Falcon®, catalog number: 305111 )
V bottom 96 well Assay plate (Corning, Costar® , catalog number: 3897 )
Glass slides Frosted (Thermo Fisher Scientific, catalog number: 12-550-11 )
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bethunaickan, R. and Davidson, A. (2012). Process and Analysis of Kidney Infiltrates by Flow Cytometry from Murine Lupus Nephritis. Bio-protocol 2(9): e167. DOI: 10.21769/BioProtoc.167.
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Category
Immunology > Immune cell function > General
Cell Biology > Cell-based analysis > Flow cytometry
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1,670 | https://bio-protocol.org/exchange/protocoldetail?id=1670&type=0 | # Bio-Protocol Content
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Chlorophyll Content Assay to Quantify the Level of Necrosis Induced by Different R Gene/Elicitor Combinations after Transient Expression
CH C. Jake Harris
DB David C. Baulcombe
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1670 Views: 9187
Edited by: Feng Li
Original Research Article:
The authors used this protocol in Dec 2013
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Abstract
This assay can be used to rapidly and accurately quantify levels of leaf necrosis induced after transient expression of R genes and elicitor combinations (Harris et al., 2013). It is based on the inverse correlation between level of necrosis and chlorophyll content in leaf tissue. It is adapted from the calculations described by (Strain et al., 1971).
Materials and Reagents
1.5 ml tube
Leaf discs
Note: Ensure that the leaf discs are fully submerged in the DMF solution.
N, N-Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: D4551-250 ml )
Equipment
Spectrophotometer (Spectronic Biomate3) (Thermo Fisher Scientific, catalog number: 335904P )
Glass spectrophotometer cuvettes (Sigma-Aldrich, catalog number: Z276898 )
Note: Product Z276898 has been discontinued.
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Harris, C. J. and Baulcombe, D. C. (2015). Chlorophyll Content Assay to Quantify the Level of Necrosis Induced by Different R Gene/Elicitor Combinations after Transient Expression. Bio-protocol 5(23): e1670. DOI: 10.21769/BioProtoc.1670.
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Category
Plant Science > Plant immunity > Disease symptom
Plant Science > Plant physiology > Tissue analysis
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1,671 | https://bio-protocol.org/exchange/protocoldetail?id=1671&type=0 | # Bio-Protocol Content
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Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths
Miloslava Fojtová
Petr Fajkus
Pavla Polanská
Jiří Fajkus
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1671 Views: 18304
Edited by: Tie Liu
Reviewed by: Igor Cesarino
Original Research Article:
The authors used this protocol in Apr 2015
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Abstract
Chromosome ends - telomeres - are a focus of intensive research due to their importance for the maintenance of chromosome stability. Their shortening due to incomplete replication functions as a molecular clock counting the number of cell divisions, and ultimately results in cell-cycle arrest and cellular senescence. Determination of telomere lengths is an essential approach in telomere biology for research and diagnostic applications. Terminal Restriction Fragments (TRF) analysis is the oldest approach to analyze telomere lengths and remains the “gold standard” even in current studies. This technique relies on the fact that repeated minisatellite telomeric units do not contain target sites for restriction enzymes. Consequently, telomeres remain in relatively long fragments (TRF), whereas the genomic DNA is digested into short pieces. Fragments of telomeric DNA are then visualized by hybridization with radioactively labeled telomeric probe. As TRF include besides telomeres also a short region of telomere-associated DNA up to the first restriction site, results are slightly shifted towards higher TRFs values. Therefore, the use of frequent cutters or their mixtures is recommended to minimize this difference. Moreover, by using TRF analysis it is possible to distinguish genuine (terminal) telomeres from interstitial telomeric repeats (ITR) (Richards and Ausubel, 1988). In this approach, BAL31 digestion is first applied on high molecular weight DNA. The enzyme progressively degrades linear DNA from its ends. The degraded DNA is then digested with one or more restriction enzymes and fragments are separated by gel electrophoresis. After blotting, membranes are probed with either a terminal marker sequence or telomeric sequence. Genuine TRF can be distinguished from ITR due to their progressive shortening with increasing BAL31 digestion time, while ITR are BAL31-resistant. The TRF BAL31 digestion pattern at the time zero indicates the approximate telomere lengths (Fajkus et al., 2005).
Keywords: Telomere Analysis Evaluation Terminal restriction fragments Southern hybridisation
Materials and Reagents
DNA isolated from plant or animal tissues (Note 1)
Plug molds (Bio-Rad Laboratories, catalog number: 170-3713 )
Restriction enzymes-Frequent-cutters restriction enzymes e.g. MseI (New England Biolabs, catalog number: R0525L ), AluI (New England Biolabs, catalog number: R0137S ), BstNI (New England Biolabs, catalog number: R0168S ), HaeIII (New England Biolabs, catalog number: R0108S ) , HinfI (New England Biolabs, catalog number: R0155L ), RsaI (New England Biolabs, catalog number: R0167S )
Note: Reaction buffers are supplied by the manufacturer.
BAL31 nuclease (New England Biolabs, catalog number: M0213L )
50 mM Ethylene glycol bis(2-aminoethylether)-N, N, N', N'-tetra acetic acid (EGTA) (pH 8.0) (SERVA Electrophoresis GmbH, catalog number: 11 290.02 )
0.5 M EDTA (pH 8.0) (Duchefa Biochemie, catalog number: E0511 )
Agarose for electrophoresis (SERVA Electrophoresis GmbH, catalog number: 11404.05 )
Agarose for pulsed field gel electrophoresis (PFGE) (Bio-Rad Laboratories, catalog number: 162-0138 )
Low melt agarose (Bio-Rad Laboratories, catalog number: 161-3112 )
Ethidium bromide aqueous solution 1% w/v (10 mg/ml) (SERVA Electrophoresis GmbH, catalog number: 21 251.01 )
DNA loading buffer (6x concentrated) (Thermo Fisher Scientific, catalog number: R0611 )
DNA marker for conventional agarose electrophoresis (e.g. GeneRuler 1 kb DNA Ladder) (Thermo Fisher Scientific, catalog number: SM0311 )
DNA marker for PFGE [e.g. Mid Range or Low Range PFG Marker (New England Biolabs, catalog number: N3551S or N0350S )]
0.25 M HCl (Penta Technologies, catalog number: 77232 )
0.4 M NaOH (Penta Technologies, catalog number: 71691 )
100 mM PMSF (in isopropanol) (SERVA Electrophoresis GmbH, catalog number: 32395 )
Proteinase K from Tritirachium album min. 8 DMC-U/mg (SERVA Electrophoresis GmbH, catalog number: 33752.02 )
N-Lauroylsarcosine sodium salt (Sigma-Aldrich, catalog number: L5125 )
D-mannitol (Duchefa, catalog number: M0803 )
Nylon membrane (Hybond XL) (GE Healthcare, catalog number: RPN303 S )
T4 polynucleotide kinase (New England Biolabs, catalog number: M0201L )
DecaLabel DNA Labeling kit (Life Technologies, catalog number: K0622 )
Note: Currently, it is “Thermo Fisher Scientific, catalog number: K0622”.
32P-γ-ATP [e.g., Institute of isotopes (Hungary, catalog number: FP-501 )]
32P-α-dATP (or 32P-α-dCTP) [Institute of isotopes (Hungary, catalog number: FP-203 )]
Synthetic telomeric oligonucleotide (4 telomeric repeats (CCCTAAA)4 or (TTTAGGG)4), or telomeric concatemers
NaCl
Tris-HCl (pH 8)
MgCl2
CaCl2
Glacial acetic acid
Boric acid
NaH2PO4
Na2HPO4
SDS
SSC
BAL31 nuclease buffer (see Recipes) or can be purchased (New England Biolabs, catalog number: B0213S )
50x TAE (see Recipes)
5x TBE (see Recipes)
Hybridization buffer (see Recipes)
Washing solution (see Recipes)
TE buffer (see Recipes)
TEM buffer (see Recipes)
Proteinase buffer (see Recipes)
Equipment
Apparatus for standard agarose gel electrophoresis (e.g. Bio-Rad Laboratories, AbD Serotec®)
PFGE electrophoresis apparatus; for optimal resolution resultion, use system with hexagonal electrodes (e.g. CHEF DR, Bio-Rad Laboratories, AbD Serotec® or Gene Navigator, Amersham)
Thermo block (e.g. Eppendorf, model: Thermomixer )
Vacuum concentrator (e.g. Thermo Fisher Scientific, model: SpeedVac )
Vacuum blotter (e.g. Bio-Rad Laboratories, AbD Serotec®)
Gel documentation system (e.g. R&D Systems, FujiFilm, model: LAS3000 )
Hybridization oven [e.g. HybriLinker (Analytik Jena, model: UVP )]
Phosphoimager (e.g. GE Healthcare Dharmacon, model: FLA7000 )
Software
Multi Gauge signal processing software (FujiFilm)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Fojtová, M., Fajkus, P., Polanská, P. and Fajkus, J. (2015). Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths. Bio-protocol 5(23): e1671. DOI: 10.21769/BioProtoc.1671.
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Category
Plant Science > Plant molecular biology > Chromatin
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1,672 | https://bio-protocol.org/exchange/protocoldetail?id=1672&type=0 | # Bio-Protocol Content
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Assay of the Carboxylase Activity of Rubisco from Chlamydomonas reinhardtii
Hemanth P. K. Sudhani
María Jesús García-Murria
Julia Marín-Navarro
Carlos García-Ferris
Lola Peñarrubia
Joaquín Moreno
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1672 Views: 9328
Edited by: Maria Sinetova
Reviewed by: Agnieszka Zienkiewicz
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
The performance of the carbon-fixing enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco), controls biomass accumulation in green plants, algae and most autotrophic bacteria. In particular, the carboxylase activity of Rubisco incorporates carbon from CO2 to ribulose 1, 5-bisphosphate (RuBP) producing two molecules of 3-phosphoglycerate. Here a detailed protocol is given for the assay of the carboxylase activity of Rubisco from Chlamydomonas reinhardtii, a model organism for chloroplast studies and a fitting host for biotechnologically oriented genetic manipulation of the enzyme. Rubisco has to be pre-incubated with Mg2+ ions and bicarbonate to induce the catalytically competent active center (Laing and Christeller, 1976). Once Rubisco is activated, the assay of its carboxylase activity described here is based on the fixation of 14C-carbon dioxide/bicarbonate into acid-resistant radioactivity (Lorimer et al., 1977). Although a spectrophotometric assay is also available (Lilley and Walker, 1974), the method based on fixation of a radioactive substrate is irreplaceable when processing a large number of samples, and it is still the technique most often used for the determination of Rubisco activity.
Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase Carbon dioxide fixation Radioisotopic assay Chlamydomonas reinhardtii Rubisco
Materials and Reagents
Sephadex G-25 columns (PD-10) (GE Healthcare, catalog number: 17-0851-01 )
Snap cap plastic tubes of 4 ml (Bio-Vials) (Beckman Coulter, catalog number: 566353 ) and tube racks
Laboratory film (Parafilm M) (Thomas Scientific, Pechiney Plastic Packaging, catalog number: PM-996 )
Chlamydomonas reinhardtii cell extract containing Rubisco
Tris [2-amino, 2-hydroxymethyl, 1, 3-propanediol] (Trizma base) (Sigma-Aldrich, catalog number: T-1503 )
Magnesium chloride hexahydrate (MgCl2.6H2O) (Merck Millipore Corporation, catalog number: 1.05833 )
Sodium bicarbonate (NaHCO3) (Merck Millipore Corporation, catalog number: 1.06329 )
D-Ribulose 1, 5-bisphosphate sodium salt hydrate (Sigma-Aldrich, catalog number: R-0878 )
Note: this is presented as a sodium salt hydrate, containing 4 Na+ and 3 H2O per molecule, with a global weight of 452.1 g/mol.
Sodium 14C-bicarbonate (52 mCi/mmol) (PerkinElmer, catalog number: NEC086H )
2, 5-diphenyl oxazol (PPO) (Sigma-Aldrich, Fluka, catalog number: 43140 )
1, 4-bis-(5-phenyl-2-oxazolyl) benzene (POPOP) (Sigma-Aldrich, Fluka, catalog number: 15150 )
Note: Product 15150 has been discontinued.
2-Phenylethylamine (Merck Millipore Corporation, catalog number: 8.07334 )
Toluene (Panreac, catalog number: 131745-1611 )
Methanol (Scharlau S.L., catalog number: ME-0301 )
Scintillation cocktail (Cocktail 22 Normascint) (Scharlau S.L., catalog number: CO-0135 )
dH2O (deionized water) (processed by the Milli-Q system from Merck Millipore Corporation)
Activation buffer (AB) (see Recipes)
Reaction buffer (RB) (see Recipes)
RuBP stock (see Recipes)
Alkaline scintillation cocktail (see Recipes)
Radioactive stock (see Recipes)
2 M Hydrochloric Acid (HCl) (VWR International, J.T. Baker®, catalog number: 6081 ) (see Recipes)
Equipment
Radioactivity licensed laboratory equipped with a security extraction hood
Thermostatic water bath (SBS, model: TI-03 )
Stopwatch chronometer (Oregon Scientific Inc., model: TR118 )
Vacuum oven (Thermo Fisher Scientific, Heraeus, model: Vacutherm VT6025 ) connected to an alkaline trap
Radioactivity scintillation counter (PerkinElmer, model: Tri-Carb 2810 TR )
Procedure
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Category
Plant Science > Phycology > Protein
Plant Science > Plant biochemistry > Protein
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1,673 | https://bio-protocol.org/exchange/protocoldetail?id=1673&type=0 | # Bio-Protocol Content
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Peer-reviewed
Purification of Rubisco from Chlamydomonas reinhardtii
Hemanth P. K. Sudhani
María Jesús García-Murria
Julia Marín-Navarro
Carlos García-Ferris
Lola Peñarrubia
Joaquín Moreno
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1673 Views: 12181
Edited by: Maria Sinetova
Reviewed by: Agnieszka Zienkiewicz
Original Research Article:
The authors used this protocol in Feb 2015
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Feb 2015
Abstract
Chlamydomonas reinhardtii is a model organism for chloroplast studies. Besides other convenient features, the feasibility of chloroplast genome transformation distinguishes this unicellular alga as ideal for the manipulation of chloroplastic gene expression aiming biotechnological goals, such as improved biofuel and biomass production. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) is the photosynthetic carbon-fixing enzyme which is considered crucial for biomass accumulation in algal cultures. Purification of wild type and site-directed mutants of Rubisco in C. reinhardtii is usually performed to study its catalytic properties and assess the carbon-fixing potential of the strains. In this protocol Rubisco is extracted through sonication of cell pellets, and purified by ammonium sulfate precipitation, sucrose gradient centrifugation (Goldwaithe and Bogorad, 1975) and anion exchange chromatography.
Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase Protein purification Chlamydomonas reinhardtii Rubisco Density gradient centrifugation
Materials and Reagents
Vacuum driven disposable filtration system (Stericup 0.22 μm) (Merck Millipore Corporation, catalog number: P25628X1 )
Graduated cylinder (50 ml)
Sephadex G-25 columns (PD-10) (GE Healthcare, catalog number: 17-0851-01 )
Glass rod
Thick wall polycarbonate plastic tubes for ultracentrifugation (Beckman Coulter, catalog number: 355631 )
Glass disposable micro pipet (Corning Inc., catalog number: 7099S-20 )
Resource Q anion exchange column 6 ml (GE Healthcare, catalog number: 17-1179-01 )
Chlamydomonas reinhardtii cells (about 8 g of wet weight collected from 2-3 liters of a 1.2 x 107 cells/ml photo-heterotrophic culture)
Tris (2-amino, 2-hydroxymethyl, 1, 3-propanediol) (Trizma base) (Sigma-Aldrich, catalog number: T-1503 )
Magnesium sulfate heptahydrate (MgSO4.7H2O) (vidraFOC, Panreac, catalog number: 131404-1210 )
Magnesium chloride hexahydrate (MgCl2.6H2O) (Merck Millipore Corporation, catalog number: 1.05833 )
Sodium chloride (NaCl) (AppliChem GmbH, Panreac, catalog number: 121659.1211 )
Sodium bicarbonate (NaHCO3) (Merck Millipore Corporation, catalog number: 1.06329 )
2-mercaptoethanol (Merck Millipore Corporation, catalog number: 8.05740 )
Sulfuric acid (H2SO4) (Sigma-Aldrich, Fluka, catalog number: 84718 )
Hydrochloric acid (HCl) (VWR International, J.T.Baker®, catalog number: 6081 )
Sucrose (Sigma-Aldrich, Fluka, catalog number: 84100 )
cOmplete (Protease inhibitors tablets) (Roche Diagnostics, catalog number: 11697 498001 )
Poly(vinylpolypyrrolidone) (Sigma-Aldrich, catalog number: P-6755 )
Ammonium sulfate [(NH4)2SO4] (AppliChem GmbH, Panreac, catalog number: 141140 )
dH2O (deionized water) (processed by the Milli-Q system from Merck Millipore Corporation)
Coomassie Blue-stained SDS-PAGE (optional)
Extraction buffer (EB) (see Recipes)
Sucrose gradient buffer (SGB) (see Recipes)
Sucrose density gradient (see Recipes)
Chromatography buffer A (CBA) (see Recipes)
Chromatography buffer B (CBB) (see Recipes)
Activation buffer (AB) (see Recipes)
Equipment
Ultrasonic processor (Sonics Vibracell, model: VCX 500 ) equipped with a 19 mm high-gain probe
Optical microscope (Microscope Central, Nikon, model: Alphaphot 2 YS2 )
Magnetic stirrer (Bibby Scientific, Stuart, model: SB161-3 ) and magnetic bars
Preparative centrifuge (GMI, Beckman Coulter, model: J2-HS ) equipped with a fixed-angle rotor (Beckman Coulter, model: JA-20 )
Ultracentrifuge (GMI, Beckman Coulter, model: L-70 ) equipped with a fixed-angle rotor (Beckman Coulter, model: 55.2 Ti )
Two-chamber (2 x 8 ml) gradient mixer (homemade)
UV Monitor (GE Healthcare, Amersham-Pharmacia Biotech, model: UV-1 with Rec112 register )
Fast Performance Liquid Chromatography equipment (FPLC) (GE Healthcare, Amersham-Pharmacia Biotech, model: Äkta Prime )
Peristaltic pump (GE Healthcare, Pharmacia, model: P-1 )
UV-V spectrophotometer (Shimadzu Scientific Instruments, model: UV-1603 )
Water jet vacuum pump (aspirator) (KARTELL SPA VIA DELLE INDUSTRIE, model: 1395 )
Ultrasonic water bath (Scientific Support, Branson, model: 2200 )
Procedure
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Category
Plant Science > Phycology > Protein
Plant Science > Plant biochemistry > Protein
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1,674 | https://bio-protocol.org/exchange/protocoldetail?id=1674&type=0 | # Bio-Protocol Content
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Peer-reviewed
Human Blood Component Vaccinia Virus Neutralization Assay
Laura Evgin
John Bell
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1674 Views: 8339
Edited by: Lee-Hwa Tai
Reviewed by: Elizabeth V. Clarke
Original Research Article:
The authors used this protocol in Jun 2015
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Jun 2015
Abstract
Many therapeutic viruses, such as oncolytic viruses, vaccines, or gene therapy vectors, may be administered by the intravenous route to maximize their delivery to target tissues. Blood components, such as antibody, complement and blood cells (such as neutrophils, monocytes, T cells, B cells or platelets) may result in viral neutralization and therefore reduce the therapeutic efficacy. This protocol will describe an in vitro assay by which to test the interaction of viruses with blood components. The effect of various factors can be isolated through fractionation. While whole blood can offer the most physiologically relevant snapshot, plasma can investigate the effects of antibody in concert with complement, and heat inactivated plasma will interrogate the effect of antibody alone.
Keywords: Complement Antibody Neutralization Virus
Materials and Reagents
BD Vacutainer 7 ml Glass Serum Tubes, No Additive, Silicone-Coated Interior (BD, catalog number: 366431 )
Eppendorf tubes (1.5 ml)
T175 cm2 culture flasks (Corning Inc., catalog number: 431079 )
12-well plates culture plates (Corning Inc., catalog number: 3513 )
15 ml Falcon tubes (BD Biosciences, Falcon®, catalog number: 352096 )
Note: Currently, it is “Thermo Fisher Scientific, FalconTM, catalog number: 352096 ”.
Insulin syringes 27G1/2” (Terumo Medical Corporation, catalog number: SS05M2713 )
0.22 μm Millipore® StericupTM filter unit (Merck Millipore Corporation, catalog number: SCGPU05RE )
Alcohol wipes
U2OS human osteosarcoma tumor cells (ATCC), grown in complete DMEM (10% FBS)
Vaccinia virus stock ≥ 5 x 106 pfu/ml
1x sterile phosphate-buffered saline (PBS) (Hyclone, catalog number: 21-031-CV )
Note: Currently, it is “Mediatech, Inc., Corning, catalog number: 21-031-CV ”.
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F1051 )
Dulbecco’s modified Eagle medium (DMEM) culture media (Hyclone, catalog number: 10-031-CV )
Note: Currently, it is “Mediatech, Inc., Corning, catalog number: 10-031-CV ”.
2x DMEM powder (Life Technologies, catalog number: 12800-017 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 12800-017 ”.
Carboxymethylcellulose (CMC) (Sigma-Aldrich, catalog number: C5678-1KG )
Sodium bicarbonate (Thermo Fisher Scientific, catalog number: S233-500 )
100x Penicillin-Streptomycin (GE Healthcare, Hyclone, catalog number: SV30010 )
Refludan (Lepirudin)
Crystal violet (Sigma-Aldrich, catalog number: C0775 )
Methanol (Thermo Fisher Scientific, catalog number: A412P-4 )
2x DMEM solution (see Recipes)
3% Carboxymethylcellulose (CMC) (see Recipes)
Titration overlay media (see Recipes)
Crystal violet (see Recipes)
Equipment
Centrifuge (Thermo Fisher Scientific, model: ST40R )
Biosafety cabinet
37 °C, 5% CO2 incubator (Sanyo)
ViCell (Beckman Coulter) or hemocytometer cell counter
pH meter
Heat blocks set to 37 °C and 56 °C
BD Vacutainer® Push Button Blood Collection Set with Pre-Attached Holder (example 25G x 0.75 in) (BD, catalog number: 367336 )
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Evgin, L. and Bell, J. (2015). Human Blood Component Vaccinia Virus Neutralization Assay. Bio-protocol 5(23): e1674. DOI: 10.21769/BioProtoc.1674.
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Category
Cancer Biology > Tumor immunology > Cancer therapy
Immunology > Complement analysis > Virus
Immunology > Host defense > Human
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1,675 | https://bio-protocol.org/exchange/protocoldetail?id=1675&type=0 | # Bio-Protocol Content
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Peer-reviewed
The Application of Quercetin to Study the Effect of Hsp70 Silencing on Plant Virus Infection in Nicotiana benthamiana Plants
Matthaios M. Mathioudakis
Ioannis Livieratos
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1675 Views: 7963
Edited by: Zhaohui Liu
Original Research Article:
The authors used this protocol in Dec 2014
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Dec 2014
Abstract
Pepino mosaic virus (PepMV) is a mechanically-transmitted pathogen affecting tomato plants worldwide. Like with other plant viruses (Verchot, 2012), the heat shock cognate protein 70 homolog (Hsc70) was identified as an interactor of the PepMV coat protein (CP) (Mathioudakis et al., 2012). Here, we describe a pharmacological approach to silence Hsp70 in plants using quercetin (Mathioudakis et al., 2014), an Hsp70 protein expression flavonoid inhibitor (Hosokawa et al., 1990; Manwell and Heikkila 2007). In the case of Hsp70, this methodology represents a faster and easier approach than silencing of Hsp70 by reverse genetics assays, such as VIGS methodology. Fully expanded leaves of 2 to 3 weeks old Nicotiana benthamiana plants were infiltrated, using a syringe, with either quercetin (dissolved in DMSO) or DMSO (control plants). The plants were mechanically inoculated with PepMV virus inocula. The accumulation of Hsp70 and PepMV were analyzed on local leaves by immunoblot analysis 4 days post inoculation.
Materials and Reagents
1 ml syringe without a needle (BD, Nipro, catalog number: SY3 1 C100U ET )
Mortar and pestles (Carl Roth GmbH + Co., catalog number: XL96.1 and XP01.1 )
Pelet pestles (Nippon Genetics, catalog number: NG006 )
13 ml round base tubes (SARSTEDT AG & Co, catalog number: 62.515.006 )
1.5 ml microtubes (SARSTEDT AG & Co, catalog number: 72.690.001 )
Nicotiana benthamiana leaves from 2 to 3 weeks old seedlings
PepMV-Sp13 isolate infected material
Quercetin hydrate (Sigma-Aldrich, catalog number: 337951 )
Dimethyl sulfoxide (DMSO) (AppliChem GmbH, catalog number: A3009 )
10 mM Sodium carbonate (Na2CO3) (Sigma-Aldrich, Riedel-de Haen, catalog number: 31432 )
Sodium dihydrogen phosphate monohydrate (0.5 M, pH 7.0) (AppliChem GmbH, catalog number: A3559 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876 )
Potassium dihydrogen phosphate (KH2PO4) (Merck Millipore Corporation, catalog number: 104873 )
Potassium chloride (KCl) (Sigma-Aldrich, Riedel-de Haen, catalog number: 31248 )
Sodium Chloride (NaCl) (AppliChem GmbH, catalog number: A1149 )
Magnesium Chloride 6-hydrate (MgCl2) (AppliChem GmbH, catalog number: A4425 )
Silicon carbide (carborundum) 400 mesh (Sigma-Aldrich, catalog number: 357391 )
Bromophenol blue (Sigma-Aldrich, catalog number: B-8026 )
Coomassie Brilliant Blue (AppliChem GmbH, catalog number: A1092 )
Glycerol (Sigma-Aldrich, catalog number: G6279 )
Tris ultrapure (1.5 M, pH 8.8; 1 M, pH 6.8) (AppliChem GmbH, catalog number: A1086 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
30% 37.5:1 Acrylamide/Bis solution (Bio-Rad Laboratories, AbD Serotec®, catalog number: 161-0158 )
10% Ammonium persulfate (APS) (AppliChem GmbH, catalog number: A2941 )
N, N, N´, N´-tetramethylethylenediamine (TEMED) (AppliChem GmbH, catalog number: A1148 )
10% Sodium dodecyl sulfate (SDS) (AppliChem GmbH, catalog number: A2263 )
Glycine (AppliChem GmbH, catalog number: A1067 )
Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Methanol (Thermo Fisher Scientific, catalog number: M/4000/17 )
Acetic acid (Sigma-Aldrich, catalog number: 33209 )
PepMV CP polyclonal antibody (Neogen/Adgen Phytodiagnostics, catalog number: 1127-01 )
HSP70 monoclonal antibody (Enzo Life Sciences, Stressgen, catalog number: N27F3-4 )
Anti-rabbit IgG, Alkaline phosphatase-conjugated antibody (Promega Corporation, catalog number: S3731 )
Anti-mouse IgG, Alkaline phosphatase-conjugated antibody (Promega Corporation, catalog number: S3721 )
BCIP/NBP Color Development Substrate (Promega Corporation, catalog number: S3771 )
PVDF membrane, Westran Clear Signal (Thermo Fisher Scientific, Whatmann, catalog number: 10485289 )
Tween-20 (Sigma-Aldrich, catalog number: P2287 )
Phosphate buffer saline (PBS) (see Recipes)
Protein extraction buffer (see Recipes)
4x Laemmli buffer (see Recipes)
SDS-PAGE buffers: Separation and Stacking gel buffers (see Recipes)
Western Blot buffers: Running, Transfer, Wash and Detection buffers (see Recipes)
Equipment
Plant growth chamber (25 °C, light:dark =16:8 h)
Biofuge stratos highspeed table centrifuge (Thermo Fisher Scientific, Heraeus, catalog number: 75005282 )
Fixed angle rotor #3334 (Thermo Fisher Scientific, Heraeus, catalog number: 75003334 )
Fixed angle microliter rotor #3332 (Thermo Fisher Scientific, Heraeus, catalog number: 75003332 )
Mini-PROTEAN 3 Cell Electrophoresis System (Bio-Rad Laboratories, AbD Serotec®, catalog number: 165-3301 )
Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-3930 )
Power Pack Supply model 200/2.0 (Bio-Rad Laboratories, AbD Serotec®, catalog number: 165-4761 )
Western blot membranes were visualized using the Gel DocTM XR Molecular Imager & System (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-8195 EDU )
Julabo Water-bath TW12 (Sigma-Aldrich, catalog number: Z615498 )
Note: Pricing & availability is not currently available.
Software
Quantification of the band intensity as absorbance units was conducted by Quantity One analysis software (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-9600)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbe-host interactions > Virus
Plant Science > Plant immunity > Host-microbe interactions
Plant Science > Plant molecular biology > Protein
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1,676 | https://bio-protocol.org/exchange/protocoldetail?id=1676&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Rhizosphere Acidification Assay
Gaston A. Pizzio
KR Kamesh Regmi
RG Roberto Gaxiola
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1676 Views: 7764
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Apr 2015
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Apr 2015
Abstract
Plant survival depends on the ability of root systems to establish themselves in locations where water and nutrients are available for uptake and translocation (Hawes et al., 2003). Rhizosphere influences crop productivity by mediating efficient nutrient transformation, acquisition, and use (Shen et al., 2013). Rhizosphere acidification is a central mechanism for plant mineral nutrition since it contributes to nutrient solubility and the proton motive force (pmf). This pmf is generated by the plasma membrane H+-ATPases (Miller and Smith, 1996; Forde, 2000) in root epidermal and cortical cells, and is coupled to active nutrient acquisition (e.g., N, K, P). Roots are able to acidify the rhizosphere by up to two pH units compared to the surrounding bulk soil mainly through the release of protons, but also bicarbonate, organic acids and CO2. Here we present an easy and inexpensive protocol to quantify protons released to the media by the root system-a method successfully used in our recently published work (Pizzio et al., 2015).
Keywords: Root Arabidopsis Rhyzosphere Nutrient-use-efficiency Acidification
Materials and Reagents
Arabidopsis thaliana seeds (Col-0)
Eppendorf tubes (1.5 ml)
Commercial Bleach
Pipettes and tips (1 and 5 ml)
Silicon caps (or aluminum foil)
Square Petri dishes
Plastic wrap
Spatula and forceps
Flasks (200 ml)
Plastic wrap
Glass culture tubes
Tween-20 (Sigma-Aldrich, catalog number: P-1379 )
Murashige and Skoog medium (MS) (PhytoTechnology Laboratories®, catalog number: M524 )
Sucrose (VWR International, catalog number: BDH-0308 )
Potassium Hydroxide (KOH) (Thermo Fisher Scientific, catalog number: P-250 )
Agar-agar (Sigma-Aldrich, catalog number: A-1296 )
Sterile distilled water
MES hydrate (Sigma-Aldrich, catalog number: M-8250 )
Seed sterilization solution (see Recipes)
MS solid (see Recipes)
MS liquid (see Recipes)
Assay solution (see Recipes)
Equipment
Autoclave
Rotary shaker
Flow hood
Fridge (4 °C)
Plant growth chamber (Conviron, catalog number: ATC26 )
Magnetic stirrer and stirring bars
pH meter (Beckman Coulter)
Note: pH probe should be capable of measuring pH in samples with volumes <= 3 ml.
Balance
Procedure
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Copyright: © 2015 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:
Pizzio, G. A., Regmi, K. and Gaxiola, R. (2015). Rhizosphere Acidification Assay. Bio-protocol 5(23): e1676. DOI: 10.21769/BioProtoc.1676.
Pizzio, G. A., Paez-Valencia, J., Khadilkar, A. S., Regmi, K., Patron-Soberano, A., Zhang, S., Sanchez-Lares, J., Furstenau, T., Li, J., Sanchez-Gomez, C., Valencia-Mayoral, P., Yadav, U. P., Ayre, B. G. and Gaxiola, R. A. (2015). Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning. Plant Physiol 167(4): 1541-1553.
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Category
Plant Science > Plant physiology > Plant growth
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1,677 | https://bio-protocol.org/exchange/protocoldetail?id=1677&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy
Teagen D. Quilichini
AS A. Lacey Samuels
Carl J. Douglas
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1677 Views: 9542
Edited by: Tie Liu
Reviewed by: Ning Liu
Original Research Article:
The authors used this protocol in Nov 2014
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Abstract
The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. The technique described was optimized for the detection of autofluorescent components of the pollen wall, including sporopollenin and the pollen coat, and provided spatial and developmental data on the autofluorescent metabolites in anthers of wild-type and pollen wall mutant plants (Quilichini et al., 2014). The use of two-photon imaging of live, intact anthers holds potential for future studies aimed at understanding the spatial relationship between gametophytic and sporophytic tissues during pollen development and the distribution of metabolites or fluorescently-tagged proteins within developing anthers.
Materials and Reagents
Ruler with millimetre divisions
Glass bottom Petri dishes (MatTek Corporation, catalog number: P35G-1.5-14C )
Coverslips (No.1.5, 0.17 mm thick, 22 x 22) (Thermo Fisher Scientific, catalog number 12-541B )
Transfer pipettes 1.5 ml
Arabidopsis thaliana plants in the flowering stage
Non-toxic food-grade paraffin wax maintained in a liquid state at 40 ºC in a paraffin bath (Thera-Band, catalog number: 24050 )
Distilled water
Equipment
Spectra-Physics MaiTai HP Titanium:Sapphire mode-locked pulsed laser, with tuneable wavelengths spanning 690 to 1,040 nm.
Olympus Fluoview 1000 scan head (Olympus America Inc., model: FV1000MPE ) modified by Olympus for two-photon imaging by the inclusion of a light path for the pulsed laser input and the housing for the non-descanned, multialkali side window photomultiplier tubes (PMTs).
Olympus BX61WI upright microscope
Olympus multiphoton microscope filter cube (440/40 bandpass filter) (OLYMPUS, model: FV10-MRVGR/XR 4CH NDD FILTER )
Olympus multiphoton microscope dedicated objective lens optimized for transmitting IR light (25x, 1.05 numerical aperture, 2.0 mm working distance) (OLYMPUS, model: FV10-SNPXLU )
Light tight, custom made multiphoton microscope enclosure. To build an enclosure, aluminum extruded components (Item North America) and flat-black painted fibre board can be used
Forceps for fine dissections (Electron Microscopy Sciences, catalog number: 72700-D )
Olympus stereomicroscope (magnification range of 6.3-63x) (OLYMPUS, model: SZX10 )
Software
Olympus FluoView FV10-ASW 3.01
Spectra-Physics MaiTai Control laser software
Volocity version 6.1.1 software package
Note: ImageJ 1.47v can also be used to process images.
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Quilichini, T. D., Samuels, A. L. and Douglas, C. J. (2015). Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy . Bio-protocol 5(23): e1677. DOI: 10.21769/BioProtoc.1677.
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Category
Plant Science > Plant developmental biology > Morphogenesis
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Two-photon microscopy
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1,678 | https://bio-protocol.org/exchange/protocoldetail?id=1678&type=0 | # Bio-Protocol Content
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Extraction of Intracellular and Cell Wall Proteins from Leaves and Roots of Harsh Hakea
MS Michael W. Shane
William C. Plaxton
Published: Vol 5, Iss 23, Dec 5, 2015
DOI: 10.21769/BioProtoc.1678 Views: 8924
Edited by: Arsalan Daudi
Reviewed by: Sollapura J. VishwanathKabin Xie
Original Research Article:
The authors used this protocol in Nov 2014
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Abstract
Plant proteins can be targeted to intracellular (i.e., cytosol, vacuole, organelles etc.) or extracellular (i.e., cell walls, apoplast) compartments. Dual targeting is a key mechanism with important implications for plant metabolism, growth, development and defense etc. Harsh Hakea (Hakea prostrata R.Br.) is a perennial species and member of the Proteaceae family that thrives on extremely phosphate impoverished soils of southwestern Australia. Harsh Hakea is not a common model organism, but has been widely developed for physiological and molecular/biochemical studies of the endogenous adaptations of an ‘extremophile’ plant species to abiotic stress, including low phosphorus tolerance. Tissues of Harsh Hakea contain large amounts of compounds (e.g., phenolics) that interfere with the extraction of soluble proteins. We previously optimised extraction of intracellular proteins from Harsh Hakea proteoid roots to improve soluble protein yield by at least 10-fold (Shane et al., 2013). Here, we describe the protocol for extraction and separation of intracellular from ‘loosely bound’ cell-wall proteins in Harsh Hakea.
Materials and Reagents
2 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-138 )
Dialysis tubing (12-14 kDa MWCO) (Spectra/Por, Fisher Scientific, catalog number: 08-667B )
25 mm syringe filters (0.45 μm) (Sarstedt, catalog number: 83.1826 )
Harsh Hakea leaves or roots
Dewar (MVE 20/15) with liquid N2, and ice bucket
Imidazole (Bioshop, catalog number: IMID508 )
Triton X-100 (Bioshop, catalog number: TRX777 )
Glycerol (Bioshop, catalog number: GLY002 )
Magnesium chloride (Bioshop, catalog number: MAG508 )
Polyethylene glycol 8000 (Bioshop, catalog number: PEG800 )
Phenylmethylsulfonyl fluoride (PMSF, G-Biosciences, catalog number: 786-0555 )
Thiourea (Sigma, catalog number: T-7875 )
Poly (vinylpolypyrrolidone) (PVPP, Sigma-Aldrich, catalog number: P-6755 )
Poly (vinylpyrrolidone) (Bioshop, catalog number: PVP504 )
Intracellular extraction buffer (see Recipes)
Resuspension buffer (see Recipes)
Cell wall extraction buffer (see Recipes)
Dialysis buffer (see Recipes)
Equipment
ZebaTM spin desalting columns 7K MWCO (0.5 ml) (Fisher Scientific, catalog number: 89882 )
Porcelain mortar (70 mm inner diameter) and pestle
Plastic pellet pestle (Kimble-Chase Kontes, Fisher Scientific, catalog number: K749521-0590 )
Macrosep® Advance Centrifugal Concentrators (10 kDa MWCO) (Pall Corporation, catalog number: MAP010C37 )
Microcentrifuge (Beckman Coulter, model: microfuge 22R )
PT-3100 Polytron Homogenizer (Kinematica, model: PT-3100)
Magnetic stirrer
4 °C cold room
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Shane, M. W. and Plaxton, W. (2015). Extraction of Intracellular and Cell Wall Proteins from Leaves and Roots of Harsh Hakea. Bio-protocol 5(23): e1678. DOI: 10.21769/BioProtoc.1678.
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Category
Plant Science > Plant biochemistry > Protein
Plant Science > Plant cell biology > Tissue analysis
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1,679 | https://bio-protocol.org/exchange/protocoldetail?id=1679&type=0 | # Bio-Protocol Content
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GC-MS-Based Analysis of Chloroform Extracted Suberin-Associated Root Waxes from Arabidopsis and Other Plant Species
Dylan K. Kosma
Isabel Molina
Owen Rowland
Published: Vol 5, Iss 24, Dec 20, 2015
DOI: 10.21769/BioProtoc.1679 Views: 11163
Edited by: Arsalan Daudi
Reviewed by: Ziqiang Zhu
Original Research Article:
The authors used this protocol in Oct 2014
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Abstract
The periderm and exodermis of taproots and tuberous taproots contain an extracellular lipid polymer, suberin, deposited in their cell walls. This polymer is intractable in organic solvents, and is co-deposited with chloroform-extractable waxes. These suberin-associated root waxes are typically composed of alkanes, primary alcohols, fatty acids, alkyl ferulates, alkyl caffeates, and alkyl coumarates (Espelie et al., 1980; Li et al., 2007; Kosma et al., 2015). They are believed to contribute to the diffusion barrier properties of suberized cell walls (Soliday et al., 1979), and possibly have other roles yet to be discovered. Here we describe a protocol to extract and analyze waxes associated with root suberin. This fraction of aliphatic components is extracted by whole root immersion in chloroform, and is then chemically modified to prepare samples that are more suitable to gas-chromatography analysis. This protocol is optimized for Arabidopsis thaliana, but can be used with roots of other plants as described herein.
Materials and Reagents
3.5 inch pots for Arabidopsis, bigger pots if necessary for other plants
9 ml glass tubes with polytetrafluoroethylene (PFTE)-lined caps (Corning, catalog number: 9826-13 )
Glass Pasteur pipets
Glass wool (Sigma-Aldrich, catalog number: 18421 )
2 ml GC vials and caps (Agilent Technologies, catalog number: 5190-2240 ) with 250 µl glass inserts (Agilent Technologies, catalog number: 5181-1270 )
10 µl and 500 µl Hamilton syringes (Hamilton Company, catalog number: 80075 and 81216 , respectively)
Note: It is advisable to rinse the glass Hamilton syringes with acetone then chloroform before each use and chloroform then acetone after each use. This will help to prevent clogging of the syringes.
Arabidopsis thaliana or other plant seeds [e.g., Brassica napus, Raphanus sativus, Beta vulgaris, see Kosma et al., (2015) for a comprehensive list of species that have been analyzed with this protocol]
Soil-less growing medium
2:1 (v/v) mixture of potting mix (Pro-Mix BX, Premier Tech Horticulture, Rivière-du-Loup)
calcined clay granules (PPC Greens Grade, Profile, Buffalo Grove)
Note: For images of these growing media please refer to https://ag.purdue.edu/hla/Hort/greenhouse/pages/101-ways-to-grow-arabidopsis.aspx
Distilled water
Chloroform (CHCl3) (≥ 99.5% purity) (Sigma-Aldrich, catalog number: C2432 )
95% Ethanol (e.g., Corning, Koptec, catalog number: V1105 )
Suggested internal standards
Pentadecanoic acid (15:0) (Nu-Check Prep, catalog number: N-15-A ) or heptadecanoic acid (17:0) (Nu-Check Prep, catalog number: N-17-A )
Tricosan-1-ol (23:0-OH) (Nu-Check Prep, catalog number: A-624 )
Monoheptadecanoin (17:0 monoacylglycerol) (Nu-Check Prep, catalog number: M-159 )
Octacosane (28:0) (Sigma-Aldrich, catalog number: O504 )
Tridecyl (13:0) ferulate, if used need to synthesize according to Kosma et al., (2012)
Heptadecyl (17:0) coumarate, if used need to synthesize according to Kosma et al., (2012)
Nonadecyl (19:0) caffeate, if used need to synthesize according to Razeq et al., (2014)
Nitrogen gas (> 99% Purity)
Pyridine, anhydrous (C5H5N) (Sigma-Aldrich, catalog number: 270970 )
N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (Sigma-Aldrich, catalog number: 15222 )
n-heptane, anhydrous (Sigma-Aldrich, catalog number: 246654 )
Toluene, anhydrous (Sigma-Aldrich, catalog number: 244511 )
Equipment
Controlled-environment plant growth chamber (e.g., Percival Scientific, Conviron, etc.)
Small pair of scissors or a razor blade
Temperature-controlled evaporator connected to nitrogen tank (e.g., Organomation Associates, catalog number: 11155 )
Dry heating block capable of reaching a temperature of 110 ºC and with a metal block containing 13 mm orifices
Gas chromatograph-mass spectrometer (e.g., Agilent Technologies, model: 6850/5975 GC-MS ) equipped with a HP-5MS capillary column (length 30 m, id 0.25 mm, film thickness 0.25 µm) (Agilent Technologies, J&W Scientific)
Software
ImageJ software (National Institute of Health)
Excel software (Microsoft office)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kosma, D. K., Molina, I. and Rowland, O. (2015). GC-MS-Based Analysis of Chloroform Extracted Suberin-Associated Root Waxes from Arabidopsis and Other Plant Species. Bio-protocol 5(24): e1679. DOI: 10.21769/BioProtoc.1679.
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Category
Plant Science > Plant biochemistry > Lipid
Plant Science > Plant metabolism > Metabolite profiling
Plant Science > Plant physiology > Tissue analysis
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168 | https://bio-protocol.org/exchange/protocoldetail?id=168&type=0 | # Bio-Protocol Content
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Isolation of Dendritic Cells and Macrophages from the Murine Kidneys of Lupus by Cell Sorter
Ramalingam Bethunaickan
AD Anne Davidson
Published: Vol 2, Iss 8, Apr 20, 2012
DOI: 10.21769/BioProtoc.168 Views: 25071
Original Research Article:
The authors used this protocol in Apr 2011
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Abstract
Methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE are essential to understand the patho-physiology of the disease. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans.An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that lead to renal impairment in SLE and other renal inflammatory diseases.
Materials and Reagents
Fetal bovine serum (FBS)
Sterile PBS (Life Technologies, Invitrogen™, catalog number: 20012-027 )
Ammonium chloride
Collagenase Type I (CLS I) (Worthington, catalog number: 4197 , specific activity 230 U mg-1)
DMEM, High glucose (Life Technologies, Gibco®, catalog number: 10313 )
Paraformaldehyde (Tousimis, catalog number: 1108A )
BSA (Fraction V) (Sigma-Aldrich, catalog number: A-7030 )
FACS antibodies:
FC block (BD Biosciences, Falcon®, catalog number: 553142 )
CD11B APC (BD Biosciences, Falcon®, catalog number: 553310 )
F/480 FITC (Serotech Laboratories, catalog number: MCA497FB )
Streptavidin PERCP (BD Biosciences, Falcon®, catalog number: 554064 )
CD4, PE (BD Biosciences, Falcon®, catalog number: 553049 )
CD5, PE (eBioscience, catalog number: 12-0051-82 )
B220, PE (BD Biosciences, Falcon®, catalog number: 553090 )
CD49b, PE (BD Biosciences, Falcon®, catalog number: 558759 )
CD11C (Biotin, catalog number: 553800 )
Streptavidin PERCP (BD Biosciences, Falcon®, catalog number: 554064)
DAPI (MP Biomedicals, catalog number: 157574 )
FACS staining buffer (see Recipes)
Equipment
FACS Aria or similar cell sorter
Bench-top refrigerated centrifuge
BD cell strainer (40 nm) (BD Biosciences, Falcon®, catalog number: 352340 )
Conical tube
30 ml syringe (BD Biosciences, Falcon®, catalog number: 309661 )
21G Needles (BD Biosciences, catalog number: 305165 )
26G needles (BD Biosciences, catalog number: 305111 )
V bottom 96 well assay plate (Corning, Costar®, catalog number: 3897 )
Glass slides frosted (Thermo Fisher Scientific, catalog number: 12-550-11 )
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bethunaickan, R. and Davidson, A. (2012). Isolation of Dendritic Cells and Macrophages from the Murine Kidneys of Lupus by Cell Sorter. Bio-protocol 2(8): e168. DOI: 10.21769/BioProtoc.168.
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Category
Immunology > Immune cell isolation > Macrophage
Cell Biology > Cell-based analysis > Flow cytometry
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1,680 | https://bio-protocol.org/exchange/protocoldetail?id=1680&type=0 | # Bio-Protocol Content
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Insertional Mutagenesis of Chlamydomonas reinhardtii
CT Chia-Hong Tsai
CB Christoph Benning
Published: Vol 5, Iss 24, Dec 20, 2015
DOI: 10.21769/BioProtoc.1680 Views: 8251
Edited by: Maria Sinetova
Reviewed by: Igor Cesarino
Original Research Article:
The authors used this protocol in Nov 2014
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Abstract
The unicellular microalga Chlamydomonas reinhardtii (C. reinhardtii) has been used as a reference model for numerous fields of research. Principle research areas are eukaryotic flagellar structure and function, basal bodies (centrioles), cell-cell recognition, cell cycle control, chloroplast biogenesis, phototaxis, nonphotochemical quenching, and especially photosynthesis for C. reinhardtii can grow in the dark on an organic carbon (e.g. acetate), and thus provides advantages over land plants (Harris, 2001; Peers et al., 2009). C. reinhardtii has a short life cycle, a sequenced genome (Merchant et al., 2007), and a growing molecular toolbox for forward and reverse genetic studies, including transformation protocols, gene silencing (Kim and Cerutti, 2009; Molnar et al., 2009), and fluorescent protein-tag (Rasala et al., 2013). There are two commonly used methods for C. reinhardtii transformation – electroporation and glass bead agitation. Electroporation is normally restricted to strains with cell wall, as it kills cell-wall-deficient strains effectively if without careful handling of osmosis. Electroporation also requires special instruments such as electroporator and cuvettes. In contrast, glass bead agitation uses simple lab equipment. The mild shear created by agitation in the presence of glass bead allows cell-wall-deficient strains to take up DNA. If glass bead method is to be applied to cell-wall strains, cells need to be treated with autolysin (http://www.chlamy.org/methods/autolysin.html) to partially lyse the wall components. A pitfall of both methods is that the DNAs are often shortened by nuclease once entering the cells, making the downstream PCR-based genotyping of insertion site rather difficult. Here I describe an improved design of insertional mutagenesis used in (Tsai et al., 2014), and the transformation protocol using glass bead as previously described in (Kindle, 1990) with minor modification. The putative mutants can be selected by autotrophic or antibiotic resistance markers, and the disrupted loci can be mapped by methods such as plasmid rescue (Peers et al., 2009) and SiteFinding PCR (Tan et al., 2005).
Keywords: Chlamydomonas Insertional mutagenesis Transformation
Materials and Reagents
15 and 50 ml conical tubes
Petri dishes (90 mm in diameter)
pHYG3 plasmid (http://chlamycollection.org/plasmid/phyg3/) or plasmid carrying other selection markers
C. reinhardtii cell wall-less strain such as dw15 (cw15, nit1, mt+; http://chlamycollection.org/strain/cc-4619-cw15-nit1-mt-dw15-1/)
Agar (Caisson Laboratories, Phytoblend)
TAP medium (Harris, 1989)
Restriction enzyme for linearizing the plasmid DNA, such as PvuII (New England Biolabs)
Hygromycin (Life Technologies) or other selection means
TAP agar plates (see Recipes)
Top agar (see Recipes)
Equipment
Common bench-top vortexer
Spectrophotometer for optical density (OD) measurement, Z2 Coulter Counter (Beckman Coulter) or a hemocytometer
250 ml flask
Bench-top centrifuge capable of centrifugation at 1,500 x g and accommodating 50 ml conical tubes
Shaker
Z2 Coulter Counter or hemocytometer
Glass beads 425-600 μm (Sigma-Aldrich, catalog number: G8772 )
Glass tubes or round bottom plastic tubes with or without cap (~10 mm in diameter)
Procedure
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Tsai, C. and Benning, C. (2015). Insertional Mutagenesis of Chlamydomonas reinhardtii. Bio-protocol 5(24): e1680. DOI: 10.21769/BioProtoc.1680.
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Category
Plant Science > Phycology > DNA
Molecular Biology > DNA > Mutagenesis
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