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28 | https://bio-protocol.org/exchange/protocoldetail?id=28&type=1 | # Bio-Protocol Content
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siRNA Transfection of Mouse Bone Marrow-derived Macrophages
RC Ran Chen
Published: Feb 5, 2011
DOI: 10.21769/BioProtoc.28 Views: 24084
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Abstract
Short interfering RNAs (siRNA) are a type of double-stranded RNA molecule, typically 20-25 bp long, that are involved in the phenomenon of RNA interference. siRNA transfection is employed in this protocol to knockdown target gene expression in BMM’phi’ cells. Two days after transfection with cells at 60-80% confluence, the knockdown efficiency can reach 90%.
Keywords: Macrophage Transfection SiRNA
Materials and Reagents
RPMI 1640 medium (RPMI) (Life Technologies, InvitrogenTM, catalog number: 11875-093 )
Fetal bovine serum (Atlanta Biologicals, catalog number: S10350 )
Stock penicillin/streptomycin (P/S) (Life Technologies, InvitrogenTM, catalog number: 15140-122 )
Lipofectamine RNAiMAX (iMAX) (Life Technologies, InvitrogenTM, catalog number: 13778150 )
Equipment
Cell counter
6-well plate
24-well plate
Procedure
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Category
Immunology > Immune cell function > Macrophage
Molecular Biology > RNA > Transfection
Molecular Biology > RNA > RNA interference
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280 | https://bio-protocol.org/exchange/protocoldetail?id=280&type=0 | # Bio-Protocol Content
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KMnO4 Footprinting
ÜP Ümit Pul
RW Reinhild Wurm
RW Rolf Wagner
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.280 Views: 26763
Original Research Article:
The authors used this protocol in Mar 2012
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Mar 2012
Abstract
The KMnO4 footprinting method offers a rapid and easy way to detect and localize single-stranded regions within a duplex DNA molecule, such as it occurs for instance within an actively transcribing RNA polymerase-DNA complex or during R-loop formation in DNA-RNA hybrid structures. The method is based on the selective oxidation of single-stranded thymines in DNA. The modified nucleotides react with strong bases by ring opening and subsequent phosphodiester cleavage. Because the modified nucleotides will not be recognized by DNA polymerase sites of modification can also be analyzed by primer extension with Klenow DNA polymerase, which stops elongation one residue before the modification. Hence, localization of the modified base positions can be performed on denaturing polyacrylamide gels either after piperidine catalyzed phosphodiester cleavage of 3'- or 5'-32P-end-labeled DNA or by primer extension with non-labeled DNA employing 32P-labeled oligonucleotide primers. Due to the fact that KMnO4 can penetrate through membranes the footprinting method can also be used for footprint analyses within living cells.
Keywords: Single-stranded DNA localization Single-stranded thymine modification DNA footprinting RNA-DNA hybrid analysis Structural analysis of DNA
Materials and Reagents
Radiolabeled DNA fragment of interest
14.3 M β-mercaptoethanol
500 mM EDTA
Phenol
Bromophenol blue (Sigma-Aldrich, catalog number: BO126 )
Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
Formamide deionized (Panreac Applichem, catalog number: A2156 )
Cholorophorm
Piperidine, purity grade: pro analysis (p.a.) (e.g. Sigma-Aldrich, catalog number: 411027 )
Ethanol (purity grade: pro analysis) (p.a.)
X-ray films
Glycogen (1 μg/μl) (e.g. Roche, catalog number: 10901393001 )
NaOAc (300 mM pH = 5.5)
Optional for Procedure B (primer extension analysis)
Non-radio labeled DNA fragment of interest
5'-32P-labeled desoxyoligonucleotide primer
NaOH (10 mM)
Tris
MgSO4
DTT
Klenow fragment of DNA polymerase I (e.g. Biolabs, catalog number: MO210S )
NH4OAc
370 mM KMnO4 stock solution (a 1:1 dilution with H2O is used for the reaction) (see Recipes)
Phenol/Chloroform (see Recipes)
Neutralization solution (see Recipes)
dNTP mix (see Recipes)
Stop mix (see Recipes)
Electrophoresis loading buffer (see Recipes)
Equipment
Table-top centrifuge
Vortex shaker
Incubator
Polyacrylamide gel electrophoresis system
Vacuum concentrator
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pul, Ü., Wurm, R. and Wagner, R. (2012). KMnO4 Footprinting. Bio-protocol 2(21): e280. DOI: 10.21769/BioProtoc.280.
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Category
Molecular Biology > DNA > DNA-protein interaction
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2,800 | https://bio-protocol.org/exchange/protocoldetail?id=2800&type=0 | # Bio-Protocol Content
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Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein
Shaarika Sarasija
KN Kenneth R. Norman
Published: Vol 8, Iss 7, Apr 5, 2018
DOI: 10.21769/BioProtoc.2800 Views: 6303
Reviewed by: Tugsan TezilJuan Facundo Rodriguez Ayala
Original Research Article:
The authors used this protocol in Dec 2015
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The authors used this protocol in:
Dec 2015
Abstract
Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), measurement of ROS levels can help us determine if mitochondrial functional integrity has been compromised. In this protocol, we describe a method to measure the level of ROS in the nematode Caenorhabditis elegans, using chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA).
Keywords: C. elegans ROS Mitochondria DCF Oxidative stress CM-H2DCFDA
Background
The life cycle of ROS is closely associated with the mitochondria. Superoxides are produced as a result of the inevitable electron leak at complex I and complex III from the electron transport chain in the mitochondria. This superoxide is then dismutated to hydrogen peroxide in the mitochondrial matrix and mitochondrial intermembrane space. The superoxide and hydrogen peroxide generated via these processes are considered to be ROS, with the mitochondria being responsible for 90% of endogenous ROS (Balaban et al., 2005). Therefore, measuring ROS levels will provide insight into the status of mitochondrial health. Here, we provide a protocol to determine the levels of ROS in the nematode C. elegans using CM-H2DCFDA, a cell-permeant chloromethyl derivative of a reduced form of fluorescein. Once inside the cell, the CM-H2DCFDA is cleaved by intracellular esterases and the resulting nonfluorescent H2DCF, upon oxidation by ROS, gets converted into the highly fluorescent 2’,7’-dichlorofluorescein (DCF). Imaging and measurement of fluorescence intensity of H2DCFDA in live C. elegans can be confounded by the presence of autofluorescence from the animal’s intestine. Our protocol utilizes worm lysates to measure ROS levels, thereby bypassing this issue (Sarasija and Norman, 2015).
Materials and Reagents
100 mm, 60 mm Petri dishes (Kord-Valmark Labware Products, catalog numbers: 2900 , 2901 )
15-ml centrifuge tubes (Globe Scientific, catalog number: 6285 )
22 x 22 mm coverslip (Globe Scientific, catalog number: 1404-10 )
Glass Pasteur pipettes (Krackeler Scientific, catalog number: 6-72050-900 )
Vacuum filtration (GE Healthcare, Whatman, catalog number: 6722-5001 )
1.5 ml Micro Centrifuge tube (CELLTREAT Scientific, catalog number: 229443 )
50 ml conical tubes (Corning, catalog number: 430829 )
15 ml conical tubes (Corning, catalog number: 430791 )
C. elegans strains and OP50 (Caenorhabditis Genetics Center (CGC), University of Minnesota)
Deionized water (dH2O)
BCA protein assay (Pierce, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23227 )
Chloro methyl-2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C6827 )
Polybead Polystyrene 0.10 μm microspheres (Polysciences, catalog number: 00876-15 )
Agarose (RPI, catalog number: A20090-500.0 )
Clear nail polish (generic)
Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-10 )
Agar (Fisher Scientific, catalog number: BP1423-2 )
Bacto peptone (BD, BactoTM, catalog number: 211677 )
Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: BP213-1 )
Potassium phosphate dibasic (K2HPO4) (Fisher Scientific, catalog number: BP363-1 )
Cholesterol (Fisher Scientific, catalog number: C314-500 )
Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285-500 )
Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-1 )
5-Fluoro-2’-deoxyuridine (FUDR) (bioWORLD, catalog number: 40690016-1 )
Bacto tryptone (BD, BactoTM, catalog number: 211705 )
Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-500 )
Sodium phosphate dibasic anhydrous (Na2HPO4) (Fisher Scientific, catalog number: BP332-1 )
Bleach (generic, plain)
Hydrogen chloride (HCl) (Fisher Scientific, catalog number: A144-500 )
Standard Nematode Growth Media (NGM) plates (see Recipes)
Sterile solutions (see Recipes)
5-Fluoro-2’-deoxyuridine containing NGM plates (see Recipes)
Sterile stocks for NGM (see Recipes)
1 M CaCl2
1 M MgSO4
1 M K2HPO4
1 M KH2PO4
1 M KPO4
LB medium (see Recipes)
M9 buffer (see Recipes)
Bleach solution (see Recipes)
10 N NaOH (see Recipes)
10x PBS (see Recipes)
Equipment
Single channel pipettes (Rainin, models: PR-10 , PR-20 , PR-200 , PR-1000 )
Finnpipette II Multichannel pipettes (Fisher Scientific, model: FisherbrandTM FinnpipetteTM II, catalog number: 21377830 )
Fisher Scientific Sonic Dismembrator with microtip probe (Fisher Scientific, model: Sonic Dismembrator Model 100 )
Thermo Electron Corporation IEC Centra CL2 Centrifuge (Thermo Fischer Scientific, Thermo ScientificTM, model: IEC Centra CL2 )
Eppendorf 5415D Centrifuge (Eppendorf, model: 5415 D )
Castle Steam Sterilizer Autoclave (Getinge, model: 433/533HC-E )
Zeiss SteREO Discovery. V8 microscope with SCHOTT Ace® I light source (ZEISS, model: SteREO Discovery.V8 )
Molecular Devices FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, model: FlexStation® 3 )
PYREX® Griffin beakers (Corning, catalog number: 1000-PACK )
PYREX® Reusable Media Storage Bottles (Fisher Scientific)
Software
SoftMax® Pro 6 Software (Molecular Devices)
Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)
GraphPad Prism 5 software package (GraphPad Software Inc., San Diego, USA)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sarasija, S. and NORMAN, K. R. (2018). Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein. Bio-protocol 8(7): e2800. DOI: 10.21769/BioProtoc.2800.
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Category
Neuroscience > Nervous system disorders > Animal model
Cell Biology > Cell metabolism > Other compound
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2,801 | https://bio-protocol.org/exchange/protocoldetail?id=2801&type=0 | # Bio-Protocol Content
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Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans
Shaarika Sarasija
KN Kenneth R. Norman
Published: Vol 8, Iss 7, Apr 5, 2018
DOI: 10.21769/BioProtoc.2801 Views: 8371
Reviewed by: Sanjib Guha
Original Research Article:
The authors used this protocol in Dec 2015
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Dec 2015
Abstract
Mitochondrial function is altered in various pathologies, highlighting the crucial role mitochondria plays in maintaining cellular homeostasis. Mitochondrial structure undergoes constant fission and fusion in response to changing cellular environment. Due to this, analyzing mitochondrial structure could provide insight into the physiological state of the cell. In this protocol, we describe a method to analyze mitochondrial structure in body wall muscles in the nematode Caenorhabditis elegans, using both transgenic and dye-based approaches.
Keywords: C. elegans Mitochondria Calcium Mitochondrial membrane potential TMRE Mitotracker
Background
itochondria are involved in ATP production, cellular respiration, calcium buffering and reactive oxidative species (ROS) metabolism (Brookes et al., 2004). Mitochondrial structure and function are dynamic and closely linked, therefore analyzing mitochondrial structure can provide clues to the status of mitochondrial health (Sarasija and Norman, 2015). We developed two sets of protocols to assess mitochondrial structure in the body wall muscle of Caenorhabditis elegans. In the first protocol, we used transgenic ccIs4251 strain in which GFP is targeted to the matrix of the body wall muscle mitochondria to visualize the mitochondria (Fire et al., 1998). In the second protocol, we used mitochondrially targeted dyes, MitoTrackerTM Red CMXRos and tetramethylrhodamine ethyl ester (TMRE) to determine the structural integrity of the body wall muscle mitochondria. Normally, animals used in in-vivo imaging are anesthetized, however anesthetizing the animals could lead to mitochondrial morphological changes (Han et al., 2012), complicating data analysis. Our protocols allow for the in-vivo imaging of mitochondrial structure in live, un-anaesthetized nematodes.
Materials and Reagents
100 mm, 60 mm Petri dishes (Kord-Valmark Labware Products, catalog numbers: 2900 , 2901 )
Glass Pasteur pipettes (Krackeler Scientific, catalog number: 6-72050-900 )
15-ml centrifuge tubes (Globe Scientific, catalog number: 6285 )
22 x 22 mm coverslip (Globe Scientific, catalog number: 1404-10 )
1.5 ml Micro Centrifuge tube (CELLTREAT Scientific, catalog number: 229443 )
50 ml conical tubes (Corning, catalog number: 430829 )
15 ml conical tubes (Corning Centristar, catalog number: 430791 )
C. elegans strains including strain SD1347, ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS + (pSAK4) myo-3p::mitochondrial GFP + dpy-20(+)] (Liu et al., 2009) and OP50 (Caenorhabditis Genetics Center (CGC), University of Minnesota)
Deionized water (dH2O)
Polybead polystyrene 0.10 μm microspheres (Polysciences, catalog number: 00876-15 )
Agarose (RPI, catalog number: A20090-500.0 )
Clear nail polish (generic)
Carl ZeissTM ImmersolTM Immersion Oil (ZEISS, catalog number: 444960-0000-000 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-10 )
Agar (Fisher Scientific, catalog number: BP1423-2 )
Bacto peptone (BD, BactoTM, catalog number: 211677 )
Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: BP213-1 )
Cholesterol (Fisher Scientific, catalog number: C314-500 )
Potassium phosphate dibasic (K2HPO4) (Fisher Scientific, catalog number: BP363-1 )
Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285-500 )
Sodium phosphate dibasic anhydrous (Na2HPO4) (Fisher Scientific, catalog number: BP332-1 )
Bleach (generic, plain)
Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-500 )
Bacto tryptone (BD, BactoTM, catalog number: 211705 )
Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
MitoTracker Red CMXRos (Thermo Fisher Scientific, InvitrogenTM, catalog number: M7512 )
Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (Thermo Fisher Scientific, InvitrogenTM, catalog number: T669 )
Standard worm (NGM) plates (see Recipes)
Sterile solutions (see Recipes)
Sterile stocks for NGM (see Recipes)
1 M CaCl2
1 M MgSO4
1 M K2HPO4
1 M KH2PO4
1 M KPO4 pH 6.0
M9 buffer (1 L) (see Recipes)
Bleach solution (see Recipes)
10 N NaOH (see Recipes)
MitotrackerTM Red CMXRos stock (see Recipes)
TMRE stock (see Recipes)
Equipment
Single channel pipettes (Rainin, models: PR-10 , PR-20 , PR-200 , PR-1000 )
Finnpipette II Multichannel pipettes (Fisher Scientific, model: FisherbrandTM FinnpipetteTM II, catalog number: 21377830 )
20 °C Incubator (Percival Scientific, model: I-41NL )
Centrifuges (Eppendorf, models: 5415 D , 5415 R ; Thermo Fischer Scientific, Thermo ScientificTM, model: IEC Centra CL2 )
Zeiss SteREO Discovery.V8 microscope with SCHOTT Ace® I light source for maintaining (ZEISS, model: SteREO Discovery.V8 )
Zeiss SteREO Discovery.V12 microscope with SCHOTT Ace® I light source and X-Cite® Series 120 Fluorescence Illuminator for transgenic selection (ZEISS, model: SteREO Discovery.V12 )
Zeiss AxioObserver microscope with Andor Clara CCD camera and X-Cite® Series 120 Fluorescence Illuminator for imaging (ZEISS, model: Axio Observer )
PYREX® Griffin beakers (Corning, catalog number: 1000-PACK )
PYREX® Reusable Media Storage Bottles (Fisher Scientific)
Software
MetaMorph® Microscopy Automation & Image Analysis Software (Molecular Devices)
ImageJ (https://imagej.nih.gov/ij/)
Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)
GraphPad Prism software package (GraphPad Software Inc., San Diego, USA)
Part I: Determining mitochondrial structure using transgenic lines
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sarasija, S. and NORMAN, K. R. (2018). Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans. Bio-protocol 8(7): e2801. DOI: 10.21769/BioProtoc.2801.
Download Citation in RIS Format
Category
Cell Biology > Tissue analysis > Tissue staining
Neuroscience > Nervous system disorders > Animal model
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2,802 | https://bio-protocol.org/exchange/protocoldetail?id=2802&type=0 | # Bio-Protocol Content
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Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells
JA Jorge Luis Ayala-Lujan
FR Fernando Ruiz-Perez
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2802 Views: 5735
Edited by: Modesto Redrejo-Rodriguez
Reviewed by: Jose ThekkiniathTomas Aparicio
Original Research Article:
The authors used this protocol in May 2017
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The authors used this protocol in:
May 2017
Abstract
Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. The aggregative adherence fimbria II (AAF/II) is the most important adherence factor of EAEC prototype strain 042 (EAEC042) to intestinal cells. Multiple receptors for AAF/II on epithelial cells have been identified including the transmembrane signaling mucin Muc1. This protocol describes a method to measure adherence of EAEC strains to HEK293 cells expressing the Muc1 glycoprotein.
Keywords: Muc1 EAEC Adherence Mucins Glycoprotein Fimbria Hek293
Background
EAEC is an important cause of endemic and epidemic diarrheal disease worldwide. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in immunocompromised adults, travelers and food-borne outbreaks in the industrialized world, such as the large lethal outbreak caused by a Shiga toxin (Stx) type 2a-producing EAEC strain of serotype O104:H4 in Northern Europe in 2011 (Harrington et al., 2006; Rasko et al., 2011). EAEC pathogenesis is determined by the organism’s ability to adhere to intestinal cells, produce enterotoxins and cytotoxins, and ultimately to induce inflammation (Harrington et al., 2006). EAEC adherence to intestinal cells is mediated by AAF fimbrial adhesins (Czeczulin et al., 1997). To date, at least five variants of the AAF fimbriae have been described, all encoded in virulence plasmids ranging from 55 to 65 MDa (Jonsson et al., 2015). The AAF structure is comprised by a positively charged major subunit and a putative minor subunit at the tip of fimbrial structures (Berry et al., 2014).
The prototype EAEC strain 042 that exhibits the AAF/II variant has been shown to produce diarrhea in adult volunteers (Nataro et al., 1995). Clinical and laboratory data suggest that EAEC induces inflammatory enteritis, while studies using polarized T84 monolayers indicates that release of IL-8 is associated to the presence of AAF/II adhesin (Harrington et al., 2005). Although the importance of the adherence of EAEC to intestinal cells has been established, the cell receptors involved in the inflammatory response mediated by AAF fimbriae have not been fully characterized. Several receptors on epithelial cells have been identified for AAF/II including extracellular matrix (ECM) proteins such as fibronectin and laminin, and cytokeratin 8 (Farfan et al., 2008; Izquierdo et al., 2014). However, these receptors are localized on the basolateral side of intestinal cells. Thus, it is unlikely that these proteins play an important role during the initial infection with EAEC. Furthermore, it has been shown that fibronectin does not participate in the inflammatory response mediated by AAF/II (Yanez et al., 2016). We have recently found that EAEC also binds to the signaling Muc1 glycoprotein, and such binding is dependent on the sialylation of the protein (Boll et al., 2017). Mucins (MUC) are large (> 200 kDa) secreted and transmembrane glycoproteins with a high carbohydrate content (50-90% by weight) expressed by a variety of normal and malignant secretory epithelial cells (Corfield et al., 2001). Muc1 is a polymorphic transmembrane mucin-like protein that contains a large extracellular domain consisting of a glycosylated polypeptide made up of 30-100 tandem repeats of a 20-amino acid sequence, a transmembrane domain, and a cytoplasmic tail of 72 amino acids (Nath and Mukherjee, 2014). Muc1 is associated to numerous signaling pathways in malignant and inflammatory processes (Nath and Mukherjee, 2014). Our recent study shows that Muc1 is associated with the inflammatory response mediated by AAF/II. Moreover, EAEC 042 up-regulates epithelial Muc1 expression dependent on the presence of AAF (Boll et al., 2017).
The existence of multiple receptors for EAEC in intestinal cells complicates the identification and characterization of specific receptors in this cell lineage. The use of HEK293 cells, which differ from enterocytes, allows the evaluation of new potential receptors through transfection of these cells with plasmids encoding the candidate receptor. Likewise, binding assays performed with cells in suspension minimizes the binding of EAEC strains to non-abiotic surfaces. This protocol could be used to find out potential receptors for other enteropathogens. Here, we described in detail the method we previously used to visualize the adherence of EAEC to HEK293 cells transfected with a Muc1-encoding plasmid.
Materials and Reagents
Cell culture flasks (Corning, catalog number: 3275 )
20, 200 and 1,000 μl Pipette tips (Barrier Low-Retention tips, Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2149P-05-RT , 2069-HR and 2279 )
24-well plate
Circle cover glasses (Fisher Scientific, Fisherbrand, catalog number: 12-545-80 )
Tissue paper
15 ml conical culture tubes (Corning, catalog number: 430052 )
Serological pipettes (1, 5, 10, 25, 50 ml) (Corning, catalog numbers: 4012 , 4051 , 4492 , 4251 , 4501 )
Glass slides (Fisher Scientific, Fisherbrand, catalog number: 12-550-123 )
Coverslip (Fisher Scientific, Fisherbrand, catalog number: 12-543C )
Cell lines: HEK293-pcDNA3.1 and HEK293-pcDNA3.1-Muc1
HEK293 cells (ATCC, catalog number: CRL-1573 )
pcDNA3.1 (Thermo Fisher Scientific, InvitrogenTM, catalog number: V79020 )
Note: pcDNA3.1-Muc1 clone was provided by Dr. Erik P. Lillehoj (Lillehoj et al., 2002).
EAEC042 strain is available from our lab strain collection (Nataro et al., 1995)
pGFP vector (Takara Bio, Clontech, catalog number: 632370 )
LB medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10855001 )
Glycerol (Fisher Scientific, Fisherbrand, catalog number: BP229-4 )
Carbenicillin
DMEM-High glucose (no phenol red), used to culture EAEC (Thermo Fisher Scientific, GibcoTM, catalog number: 31053028 )
Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 70011044 )
Dulbecco’s modified Eagle’s medium (DMEM), used to culture HEK293 cells (Thermo Fisher Scientific, GibcoTM, catalog number: 11965118 )
Fetal bovine serum, certified, heat inactivated (FBS) (Sigma-Aldrich, catalog number: F4135 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
G418 (Sigma-Aldrich, catalog number: G418-RO)
Manufacturer: Roche Diagnostics, catalog number: 4727878001 .
0.5 M EDTA (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9260G )
Methanol (Sigma-Aldrich, catalog number: 322415-1L )
Glacial acetic acid (Fisher Scientific, Fisher Chemicals, catalog number: A38-500 )
FITC Mouse anti-Human Muc1 (CD227) (BD, BD Biosciences, catalog number: 559774 )
Coverslip sealant (Biotium, catalog number: 23005 )
FM 4-64FX (Thermo Fisher Scientific, InvitrogenTM, catalog number: F34653 )
ProLongTM Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36935 )
4’,6-Diamidino-2-Phenylindole (DAPI, Dilactate) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3571 )
Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14175103 )
Formalin (Sigma-Aldrich, catalog number: HT501128-4L )
Equipment
10, 20, 200, 1,000 μl pipettes (Pipette Pack Set, Eppendorf, catalog number: 2231300006 )
CO2 incubator (NuAire, model: NU-4750 )
-80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: Forma Model 900 )
Rotary shaker (ATR, model: AJ118 )
Laminar flow hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series A2 Model 1375 )
UV-Vis Spectrophotometer (GE Healthcare, Amersham Biosciences, model: Ultrospec 2100 Pro )
Liquid nitrogen storage container (Worthington, model: LD35 )
Centrifuge (Eppendorf, model: 5430 )
Microcentrifuge (Fisher Scientific, model: accuSpinTM Micro 17R )
Epifluorescence microscope (Olympus, model: BX51 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ayala-Lujan, J. L. and Ruiz-Perez, F. (2018). Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells. Bio-protocol 8(8): e2802. DOI: 10.21769/BioProtoc.2802.
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Category
Microbiology > Microbe-host interactions > In vitro model
Immunology > Mucosal immunology > Epithelium
Cell Biology > Cell-based analysis > Cell adhesion
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2,803 | https://bio-protocol.org/exchange/protocoldetail?id=2803&type=0 | # Bio-Protocol Content
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Peer-reviewed
Electrophysiological Recordings of Evoked End-Plate Potential on Murine Neuro-muscular Synapse Preparations
Giulia Zanetti*
SN Samuele Negro*
AM Aram Megighian
Marco Pirazzini
*Contributed equally to this work
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2803 Views: 11203
Edited by: Andrea Puhar
Reviewed by: Jackeline Moraes MalheirosAlexandros Kokotos
Original Research Article:
The authors used this protocol in Aug 2017
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Aug 2017
Abstract
Neuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure and functionality.
Electrophysiology is the most accurate experimental procedure to study NMJ physiology and it largely contributed to the elucidation of synaptic transmission basic principles. Many electrophysiological techniques have been developed to study NMJ physiology and physiopathology. In this paper, we describe an ex vivo tissue preparation for electrophysiology that can be applied to investigate nerve-muscle transmission functionality in mice. It is routinely used in our laboratory to study presynaptic neurotoxins, antitoxins, and to monitor NMJ degeneration and regeneration. This is a broadly applicable technique which can also be adopted to investigate alterations of NMJ activity in mouse models of neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.
Keywords: Neuromuscular junction Electrophysiology Evoked End-Plate Potential (eEPP) Miniature End-Plate Potential (mEPP) Neurotoxins Regeneration
Background
Neurotransmission is the physiological process by which neurons transfer information to target cells on a rapid time scale (usually < 1 msec). The structure mediating this communication is the synapse, a specialized structure formed either between neurons (a pre- and a post-synaptic neuron) or between a neuron (presynaptic neuron) and an effector cell (post-synaptic cell). Neuromuscular junction (NMJ) is the chemical synapse enabling communication between motor neuron and skeletal muscle fiber. This is the best characterized synapse and most of the knowledge on maturation, structure and function of synapses derives from its study (Li et al., 2016). At the NMJ, the action potential running along the motor axon invades the nerve terminal (presynaptic bouton) and induces the opening of voltage-gated calcium channels. The ensuing Ca2+ influx in presynaptic nerve terminal triggers (approximately in 0.3 µsec (Kuffler et al., 1984)) the fusion with the presynaptic membrane of about 100 synaptic vesicles from a ready to release pool (10-20% of all vesicles) (Del Castillo and Katz, 1954; Denker and Rizzoli, 2010). Around 1,000 acetylcholine (ACh) molecules per vesicle diffuse in the synaptic cleft (Kuffler and Yoshikami, 1975) and, in about 0.5 msec, bind to the nicotinic ACh Receptors (nAChRs) on the postsynaptic muscle fiber membrane. nAChRs are ionotropic ligand-gated Na+/K+ channels which open upon ACh binding and cause a local depolarizing potential of the postsynaptic membrane (end-plate) by mediating a large inward flux of Na+ (and a smaller outward flow of K+). This local depolarization is named evoked End-Plate Potential (eEPP) (or Evoked Junction Potential). In mice, the resting membrane potential of a skeletal muscle fiber lies around -75 mV and the eEPP has an amplitude of ~15-30 mV (depending on muscle type). When the eEPP amplitude is sufficiently high to reach or overcome action potential threshold, voltage-gated Na+ channels open thus triggering an action potential into the muscle fiber, which ultimately spreads out along the sarcolemma and invades muscle fiber T-tubules. Here, an excitation-contraction molecular machinery transduces this electric signal into the cytosolic release of Ca2+ from the sarcoplasmic reticulum, leading to muscle fiber contraction (Figure 1).
Figure 1. Mechanism of muscle fiber contraction. Acetylcholine (ACh), released by synaptic vesicle (SVs) fusing with the motor axon terminal membrane, binds to post synaptic nicotinic ACh Receptors, ionotropic cation channels that upon binding allow the leakage of cations (Na+ inward, K+ outward), leading to a local depolarization of the sarcolemmatic membrane (eEPP). When depolarization is sufficiently large to overcome the voltage threshold (red dotted line), voltage gated Na+ channels get open and trigger a post synaptic action potential (AP) spreading along the sarcolemma and invading the T-tubules (invagination of the sarcolemma within the muscle fiber). Dihydropyridine (DHP) receptors sense this membrane depolarization and stimulate the opening of Ryanodine Receptors (RyR) on the sarcoplasmic reticulum which release Ca++ into the cytosol eliciting muscle contraction. µ-conotoxin inhibits voltage gated Na+ channels thus allowing to record membrane depolarization due to the opening of the sole nicotinic AChR, i.e., the eEPP.
Random fusion of synaptic vesicles also takes place in the absence of a presynaptic action potential thereby inducing a very small (~0.4 mV) depolarization of the end-plate, which is not sufficient to trigger muscle contraction. This spontaneous activity is called ‘miniature End-Plate Potential’ (mEPP) and, according to the quantal hypothesis, it is generated by the release of a single synaptic vesicle (Katz, 2003).
NMJ is easily accessible to many kinds of experimental manipulation. Since the ‘50s’ electrophysiology applied at the NMJ provided seminal discoveries on basic aspects of synaptic transmission (Augustine and Kasai, 2007). Thereafter, a continuous development in technics and animal models paved the way to sophisticate investigation of pathological alterations occurring at the NMJ in neuromuscular diseases, including myasthenic syndromes and peripheral neuropathies, as well as neuroparalytic syndromes caused by animal (Duchen et al., 1981; Duregotti et al., 2015a) and bacterial toxins (Colasante et al., 2013, Pirazzini et al., 2014).
We describe here a detailed protocol to evaluate NMJ functionality in murine muscle-nerve preparations. The method is based on the intracellular recording of spontaneous mEPPs and nerve-evoked EPPs in muscle fibers of soleus nerve-muscle preparations, thus allowing accurate investigation of NMJ functionality at a single synapse resolution (Tremblay et al., 2017). We have recently used this method to test engineered botulinum neurotoxins and to assay the efficacy of novel putative antitoxins (Pirazzini et al., 2014; Zanetti et al., 2017). In addition, we successfully employed this technique to study NMJ nerve degeneration and to test molecules promoting its regeneration (Duregotti et al., 2015b; Negro et al., 2017; Rigoni and Montecucco, 2017).
This procedure represents a basic technique that can be easily adopted to investigate NMJ activity in mouse models of any neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.
Materials and Reagents
Silver wire (World Precision Instruments, catalog number: AGW2030 )
1 ml syringe (CHEMIL s.r.l., Padova, catalog number: S01G25 )
Petri dish 35 mm (any producer is fine)
Petri dish, 35 x 10 mm, coated with Sylgard (Dow Corning, Sylgard® 184 Silicone Elastomer kit)
Flexible needle electrode Microfil (World Precision Instruments, catalog number: MF34G-5 )
Tips for 2 μl micropipette
Tips for 200 μl micropipette
Tips for 1,000 μl micropipette
Glass capillaries for intracellular microelectrodes (length 100 mm, inner diameter 0.86 mm, outer diameter 1.50 mm; Science Products, catalog number: GB150F-10 )
Glass capillaries for stimulating microelectrode (length 100 mm, inner diameter 1.05 mm, outer diameter 1.50 mm; Science Products, catalog number: GB150TF-10 )
Mice of desired strain and age
Note: We used here plp-GFP C57BL/6J transgenic mice.
Iron(III) chloride (FeCl3) (Sigma-Aldrich, catalog number: 451649 )
Silver chloride (AgCl) (Sigma-Aldrich, catalog number: 204382 )
μ-Conotoxin GIIIB (Alomone, Jerusalem, Israel)
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
Magnesium chloride, standard solution 1 M (MgCl2) (Honeywell International, Fluka, catalog number: 63020 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: H1758 )
Potassium acetate (CH3COOK) (Sigma-Aldrich, catalog number: P3542 )
Ringer’s solution (see Recipes)
Recording electrode solution (see Recipes)
Equipment
Micropipettes
Volumetric flask (typically 500 ml; any producer is fine)
Electrophysiology setup complete with antivibration table (Newport, USA) (Figure 3)
Stereomicroscope for the electrophysiology setup (Leica Microsystem, model: Leica MZ125 , numeric aperture 0.8 with plan apochromatic objective 1.6x) (#1 in Figure 3)
Hydraulic micromanipulator for intracellular recording electrode (NARISHIGE, model: MHW-103 , Three-axis Water Hydraulic Micromanipulator) (#2 in Figure 3)
Stimulation electrode micromanipulators (Manual Micromanipulator, MÄRZHÄUSER WETZLAR, model: MM 33 ) (#3 in Figure 3)
Faraday cage (#4 in Figure 3, home-made) and stimulator: S88 stimulator (Grass, Warwick, RI, USA) (#5 in Figure 3)
Amplifier: intracellular bridge mode amplifier (Npi electronic, model: BA-01X ) (#6 in Figure 3)
2 Forceps (Micro Jewelers Forceps, Rudolf Medical, catalog number: RU 4240-05 )
Scissors (Micro Spring Scissors, Rudolf Medical, VANNAS, catalog number: RU 2260-08 )
Scissors (Delicate Surgical Scissors, Rudolf Medical, catalog number: RU 1503-12 )
Dissection microscope (OPTIKA Microscopes, model: SZM-LED2 )
Pipette puller (P-97 Flaming/Brown Micropipette Puller) (Sutter Instruments, model: P-97 )
A/D interface (National Instruments, model: PCI-6221 ) and computer compatible with the software (#7 in Figure 3)
Gas tank 95% O2 with 5% CO2 (any size and any supplier are fine)
Cylinder pressure regulator (Air Liquid, model: HBS 240-1-2 )
Software
Recording: WinEDR free software (Strathclyde University, Glasgow, Scotland, UK)
Analysis: Clampfit (Molecular Devices, Sunny-vale, CA, USA)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zanetti, G., Negro, S., Megighian, A. and Pirazzini, M. (2018). Electrophysiological Recordings of Evoked End-Plate Potential on Murine Neuro-muscular Synapse Preparations. Bio-protocol 8(8): e2803. DOI: 10.21769/BioProtoc.2803.
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Category
Neuroscience > Peripheral nervous system > Skeletal muscle
Cell Biology > Cell-based analysis > Electrophysiological technique
Cell Biology > Cell signaling > Synaptic transmision
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2,804 | https://bio-protocol.org/exchange/protocoldetail?id=2804&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis
BS Benjamin Smith
Jianfang Li
MM Matteo Metruccio
Stephanie Wan
David Evans
SF Suzanne Fleiszig
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2804 Views: 6433
Edited by: Andrea Puhar
Reviewed by: Timo A LehtiAlexander B Westbye
Original Research Article:
The authors used this protocol in May 2017
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Original research article
The authors used this protocol in:
May 2017
Abstract
A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples.
Keywords: Bacteria Pseudomonas aeruginosa Twitching motility Quantification Differential interference contrast microscopy Computational image analysis
Background
Pilus-mediated twitching motility represents a form of surface-associated bacterial movement that is independent of flagella. Twitching motility is utilized by many bacterial pathogens, including Neisseria gonorrhoeae and Pseudomonas aeruginosa, to interact with moist surfaces and translocate epithelial barriers. In P. aeruginosa, twitching motility is regulated by a large number of genes which allow both extension and retraction of type IV pili to effectively drag the bacterial cell across any given surface in response to environmental cues (Mattick, 2002; Whitchurch et al., 2004; Burrows, 2005). In our studies of P. aeruginosa pathogenesis, twitching motility contributes to bacterial exit from epithelial cells after internalization and bacterial traversal of multilayered corneal epithelia (Alarcon et al., 2009). In a murine model of corneal infection, twitching motility was important for P. aeruginosa virulence (Zolfaghar et al., 2003). Recently, we discovered that the glycoprotein DMBT1 found in mucosal fluids such as human tears and saliva was capable of inhibiting P. aeruginosa twitching motility (Li et al., 2017). In that study, we utilized a novel method to quickly and robustly quantify P. aeruginosa twitching motility. That protocol is presented herein.
The most direct way to quantify twitching motility would be to track all individual bacteria over time. This method was attempted as part of our original study. However, bacterial colonies have a complex 3D structure, with bacteria regularly traversing one another, making tracking feasible only near the colony edge, resulting in sampling bias. Previous methods for quantifying twitching motility also quantified motility only at the colony edge (Alarcon et al., 2009; Semmler et al., 1999). For our study, we wished to extend those methods to be able to quantify twitching behavior throughout a dense bacterial colony. Instead of focusing on the direction of motility, we focused on quantifying the degree of motility at any given point. This turned out to be a simpler problem to solve, since as bacteria move, they modulate light as they pass through a given point. By mapping out the relative magnitudes of this modulation over time, we were able to generate maps of regions of relatively high and low motility in dense, spatially complex colonies.
Materials and Reagents
Glass slides (Fisher Scientific, Fisherbrand, catalog number: 12-550-15 )
Cover slip (Fisher Scientific, Fisherbrand, catalog number: 12-545M )
Small Petri dish (Corning, Falcon®, catalog number: 351029 )
Large Petri dish (Sigma-Aldrich, catalog number: P5981-100EA)
Manufacturer: Excel Scientific, catalog number: D-902 .
Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
Inoculation loop (Fisher Scientific, Fisherbrand, catalog number: 22-363-597 )
Sterile wooden toothpick (any brand, autoclave at 121 °C for 15 min in a foil-covered container)
Pseudomonas aeruginosa strain MPAO1 (Dr. Manoil Laboratory, University of Washington, Seattle, WA), or other piliated strain (or bacterial species) with functional twitching motility. (MPAO1 is a wild-type P. aeruginosa strain, and is available from the University of Washington, Seattle, WA. http://www.gs.washington.edu/labs/manoil/libraryindex.htm)
Substance to be tested in the assay (e.g., human tear fluid)
Deionized water
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Chemical, catalog number: M63-500 )
Gellan gum (Alfa Aesar, catalog number: J63423 )
Tryptone (BD, BactoTM, catalog number: 211705 )
Sodium chloride (NaCl) (Fisher Scientific, Fisher Chemical, catalog number: S271-3 )
Yeast extract (BD, BactoTM, catalog number: 288620 )
Trypticase Soy Agar (TSA) powder (BD, DifcoTM, catalog number: 236950 )
Twitching medium (see Recipes)
Trypticase Soy Agar (TSA) plate (see Recipes)
Equipment
Tweezers (Dumont, Dumoxel Nº5, Fine Science Tools, catalog number: 11252-30 )
Bunsen burner
3.60 °C oven (Boekel Scientific, model: 133000 )
Sterile Biosafety Cabinet (NUAIRE, model: NU-425-600 )
Compound microscope with a high NA objective and digital camera (≥ 1.2). Our study used:
Nikon Ti-E inverted wide-field fluorescence microscope (Nikon Instruments, model: Eclipse Ti-E )
Uno-combined controller (Okolab) and stage-top incubation chamber to maintain samples at 37 °C (Okolab, model: H301-Nikon-TI-S-ER )
CFI Plan Apo Lambda 60x NA 1.4 oil objective (Nikon Instrument, model: CFI Plan Apochromat Lambda (λ) Series )
DS-Qi2 Monochrome CMOS Camera (Nikon Instrument, model: DS-Qi2 )
Computer. MacPro5.1 (2012), 2x 2.4 GHz 6-Core Intel Zeon, 64 GB 1333 MHz DDR 3 ECC
Software
ImageJ (version 1.51n) or FIJI (http://fiji.sc/)
R compiler (version x64 3.4.1) (https://www.r-project.org/)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Smith, B. E., Li, J., Metruccio, M., Wan, S., Evans, D. J. and Fleiszig, S. M. J. (2018). Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis. Bio-protocol 8(8): e2804. DOI: 10.21769/BioProtoc.2804.
Download Citation in RIS Format
Category
Microbiology > Microbe-host interactions > Bacterium
Cell Biology > Cell imaging > Live-cell imaging
Cell Biology > Cell movement > Cell motility
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2,805 | https://bio-protocol.org/exchange/protocoldetail?id=2805&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
A Bioinformatics Pipeline for Whole Exome Sequencing: Overview of the Processing and Steps from Raw Data to Downstream Analysis
NM Narendra Meena
PM Praveen Mathur
KM Krishna Mohan Medicherla
Prashanth Suravajhala
Published: Apr 20, 2018
DOI: 10.21769/BioProtoc.2805 Views: 31064
Reviewed by: Beatrice Li
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Abstract
Recent advances in Next Generation Sequencing (NGS) technologies have given an impetus to find causality for rare genetic disorders. Since 2005 and aftermath of the human genome project, efforts have been made to understand the rare variants of genetic disorders. Benchmarking the bioinformatics pipeline for whole exome sequencing (WES) has always been a challenge. In this protocol, we discuss detailed steps from quality check to analysis of the variants using a WES pipeline comparing them with reposited public NGS data and survey different techniques, algorithms and software tools used during each step. We observed that variant calling performed on exome and whole genome datasets have different metrics generated when compared to variant callers, GATK and VarScan with different parameters. Furthermore, we found that VarScan with strict parameters could recover 80-85% of high quality GATK SNPs with decreased sensitivity from NGS data. We believe our protocol in the form of pipeline can be used by researchers interested in performing WES analysis for genetic diseases and any clinical phenotypes.
Keywords: Whole exome sequencing Next generation sequencing Bioinformatics pipeline Variants Genetics Clinical phenotypes
Background
Next Generation Sequencing (NGS) technologies have paved the way for rapid sequencing efforts to analyze a wide number of samples. From the whole genome to transcriptome to exome, it has changed the way we look at nonspecific germline variants, somatic mutations, structural variant besides identifying associations between a variant and human genetic disease (Singleton et al., 2011). This can help understand the complex genetic disorders to get better diagnosis and assess disease risk. The analysis of exome sequencing data to find variants, however still poses multiple challenges. For example, there are several commercial and open source pipelines but configuring (Pabinger et al., 2014; Guo et al., 2015) them in terms of benchmarking and optimizing them is a time-consuming process. Among the steps, viz. quality check, alignment, recalibration, variant calling, variant annotation, one needs to reach consensus on the set of tools following which one’s output should be fed as other tool’s input (Stajich et al., 2002; Gentleman et al., 2004; Chang and Wang, 2012). While integrating, it would be appropriate to check and use the tools before reproducing and maintaining highly heterogeneous pipelines (Hwang et al., 2015). In this protocol, we discuss the steps for whole exome sequence (WES) analyses and its pipeline to identify variants from exome sequence data. Our pipeline includes open source tools that include a number of tools from quality check to variant calling (see Software section).
Equipment
Computer
64GB RAM with 8 core CPUs in an Ubuntu operating system (14.04 LTS machine)
Software
All the software can be downloaded/used from following locations:
FastQC https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Bowtie2 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Samtools http://samtools.sourceforge.net/
VarScan http://varscan.sourceforge.net/
Bcftools https://github.com/samtools/bcftools
Vcftools https://vcftools.github.io/index.html
PANTHER http://pantherdb.org/
dbSNP https://www.ncbi.nlm.nih.gov/projects/SNP/
1000 genomes dataset http://www.internationalgenome.org/
GeneMania http://genemania.org/
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
Category
Systems Biology > Genomics > Exome capture
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2,806 | https://bio-protocol.org/exchange/protocoldetail?id=2806&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Quantification of Thrips Damage Using Ilastik and ImageJ Fiji
IV Isabella G. S. Visschers
ND Nicole M. van Dam
JP Janny L. Peters
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2806 Views: 9527
Edited by: Pengpeng Li
Reviewed by: Eugenio LlorensOliver Xiaoou Dong
Original Research Article:
The authors used this protocol in Jun 2018
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Original research article
The authors used this protocol in:
Jun 2018
Abstract
Quantification of insect damage is an essential measurement for identifying resistance in plants. In screening for host plant resistance against thrips, the total damaged leaf area is used as a criterion to determine resistance levels. Here we present an objective novel method for analyzing thrips damage on leaf disc using the freely available software programs Ilastik and ImageJ. The protocol was developed in order to screen over 40 Capsicum lines for resistance against Frankliniella occidentalis (Western Flower Thrips) and Thrips tabaci (Onion thrips).
Keywords: Insect resistance Insect damage Image analyses
Background
Quantification of insect damage is an essential measurement for identifying resistance in plants. In screening programs for host-plant resistance against thrips, the total damaged leaf area is used as a criterion to determine resistance levels. Thrips damage is characterized by silvery spots that show high contrast with the intact leaf area, but the feeding spots also include darker areas ranging from dark green to brown. These gradual discolorations of the leaf are too subtle to precisely quantify with programs such as Winfolia (http://www.regentinstruments.com/assets/winfolia_software.html) or ImageJ (Rasband, 2011) alone. As a result, thrips damage is commonly scored by individuals that rate the samples. Samples are classified into categories signifying the amount of damage (Mirnezhad et al., 2010; Maharijaya et al., 2011 and 2012), or damage is estimated to the nearest 1 mm2 (Leiss et al., 2009; Mirnezhad et al., 2010; Maharijaya et al., 2011 and 2012). These subjective measurements make comparison between studies/screening programs difficult. Moreover, they are time consuming and thus costly for breeding companies. Here we present an objective high-throughput standardized screening method to measure leaf surface damage caused by thrips using the freely available software programs ImageJ Fiji (Schindelin et al., 2012) and Ilastik (Sommer et al., 2011). Ilastik has a wide range of applications ranging from cell biology (Fabrowski et al., 2013), where it is used to compute the amount of surface flattening of epithelial cells, to biomechanics (Bongiorno et al., 2014), where it is used to identify boundaries of human mesenchymal stem cells. It is an easy-to-use, self-learning image processing program that allows segmentation and classification of two-dimensional surfaces based on labels provided by the user (Sommer et al., 2011). ImageJ is often used to quantify the amount of removed leaf area by chewing herbivores and the total leaf surface of intact leaves (Meyer and Hull-Sanders, 2008; Morrison and Lindell, 2012). However, it is rarely used to quantify feeding damage caused by thrips. Thrips feeding causes rather subtle discolorations on the leaves. ImageJ is limited in quantifying such color differences, for which Ilastik provides a more suitable alternative.
Materials and Reagents
Filtration paper (e.g., filtration paper nr 600, VWR, catalog number: 516-0309 )
Ziploc® like bags (e.g., 18 x 25 cm, 50 µm polyethylene foliezak met druksluiting, Vink Lisse, catalog number: 174718 49 )
Petri Dish diameter 15 cm (e.g., non-treated culture dishes, Corning, catalog number: 430597 )
Parafilm® M (e.g., BRAND®, Parafilm®, VWR, catalog number: 291-1213 )
Glass jar (Figure 1) (e.g., 555 ml Twist-off pot TO82 with Twist-off deksel RTS82 wit BPA NI lid, www.glazenflessenenpotten.nl, GLAZEN RLESSEN EN POTTEN. NL, catalog numbers: V2391WS and VC305 )
Figure 1. Glass jar for thrips starvation (height 11.5 cm x diameter 7.5 cm)
Synchronized L1/L2 Frankliniella occidentalis (Pergande) or Thrips tabaci (Lindeman) (Thysanoptera)
Capsicum annuum (Solanaceae) plants (any variety)
Agar (e.g., Phyto Agar, Duchefa Biochemie, catalog number: P1003 )
Water
1.5% liquid agar solution (see Recipes)
Equipment
Climate cabinet set to 25 °C for F. occidentalis or 23 °C for T. tabaci, L16:D8 light regime (e.g., Economic Delux 432 L with TL lightning, Snijders Labs, http://www.snijderslabs.com)
Microwave (Moulinex, model: Micro-chef FM2515Q )
Cork borer, diameter 1.5 cm (e.g., Humboldt Brass Cork Borer Set with Handles, Fisher Scientific, catalog number: 07-865-10B)
Manufacturer: Humboldt Mfg., catalog number: H-9663 .
Beaker 50 ml (e.g., Griffin beakers, Corning, PYREX®, catalog number: 1000-50 )
Soft paint brush (e.g., van Eyck paint brush set, brush #1)
Plastic tweezers (e.g., Azlon Forceps - Tweezers, Dynalab Corp., Dynalon Labware, catalog number: 516555-0001 , https://www.dynalon.com/PublicStore/)
Epson 10000XL scanner (Epson, model: 10000XL ) or any SLR camera (12 mega pixel) with tripod
Handmade grid with 2 cm spacing (Figure 3)
Black paper (for scanner) or black cloth (for SLR camera)
Laptop with installed software
Precision balance (Sigma-Aldrich, catalog number: Z267074)
Manufacturer: Sartorius, model: BP 310 S .
Laboratory bottle with cap 500 ml (DWK Life Sciences, Duran, catalog number: 21 801 44 5 )
Software
ImageJ Fiji e.g., version 2.0.0 with Java 1.6.0_24
Ilastik version 1.1.3,
Note: For successful application of the protocol, it is important to use this exact version. The software can be found online: files.Ilastik.org/1.1/, ‘ilastik-1.1.3-win64.exe’, also available for Linux and OSX.
Epson Scan Utility e.g., version 3.4.9.9
Note: Only necessary if an Epson scanner is used or equivalent when using another scanner.
Procedure
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How to cite：Visschers, I. G. S., van Dam, N. M. and Peters, J. L. (2018). Quantification of Thrips Damage Using Ilastik and ImageJ Fiji. Bio-protocol 8(8): e2806. DOI: 10.21769/BioProtoc.2806.
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Category
Plant Science > Plant immunity > Disease symptom
Plant Science > Plant physiology > Biotic stress
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2,807 | https://bio-protocol.org/exchange/protocoldetail?id=2807&type=0 | # Bio-Protocol Content
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Peer-reviewed
Qualitative Analysis of Lipid Peroxidation in Plants under Multiple Stress Through Schiff’s Reagent: A Histochemical Approach
Jay Prakash Awasthi
BS Bedabrata Saha
Bhaben Chowardhara
Sanjenbam Sanjibia Devi
Pankaj Borgohain
SP Sanjib Kumar Panda
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2807 Views: 13203
Edited by: Samik Bhattacharya
Reviewed by: Venkatasalam Shanmugabalaji
Original Research Article:
The authors used this protocol in Apr 2017
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Apr 2017
Abstract
Lipid peroxidation is a physiological indicator of both biotic and abiotic stress responses, hence is often used as a biomarker to assess stress-induced cell damage or death. Here we demonstrate an easy, quick and cheap staining method to assess lipid peroxidation in plant tissues. In this methodology, Schiff’s reagent, is used to assay for membrane degradation. Histochemical detection of lipid peroxidation is performed in this protocol. In brief, Schiff’s reagent detects aldehydes that originate from lipid peroxides in stressful condition. Schiff’s reagent is prepared and applied to plants tissue. After the reaction, plant tissue samples are rinsed with a sulfite solution to retain the staining color. From this analysis, qualitative visualization of lipid peroxidation in plant tissue is observed in the form of magenta coloration. This reagent is useful for visualization of stress induced lipid peroxidation in plants. In this protocol, Indica rice root, Assam tea root and Indian mustard seedlings are used for demonstration.
Keywords: Schiff’s reagent Rice Tea Mustard Membrane degradation Lipid peroxidation
Background
Oxidative stress induced by both biotic and abiotic stress factors leads to lipid peroxidation. The peroxidation of unsaturated lipids in membrane is the most apparent symptom of oxidative stress. Lipid peroxidation is a deleterious process in plants, which affects membrane properties, causes protein degradation and limits the capacity of ionic transport, ultimately triggering the cell death process (Yamamoto et al., 2001). Malondialdehyde (MDA) content, which is a byproduct of lipid peroxidation process was found to be enhanced in rice (Ma et al., 2012; Awasthi et al., 2017), Salvinia (Mandal et al., 2013), pea (Yamamoto et al., 2001), Soyabean (Cakmak and Horst, 1991; Du et al., 2010), Indian mustard (Saha et al., 2016) on exposure to stress. The reactive oxygen species (ROS) associated with oxidative stress acts on membrane lipids to decrease membrane stability. Thus making the study of lipid peroxidation an important parameter and our protocol offers a very rapid method for the study. Schiff’s reagent reacts with aldehyde functional group of MDA to give the magenta coloration, thus acting as a key determinant of lipid peroxidation in plant tissues.
Materials and Reagents
Glass Petri plates of 10 cm diameter (Scott Duran)
Absorbent cotton
Whatman 1 filter paper
Razor blade (Glassvan, No. 23, Scalpel blade)
15 ml disposable centrifuge tubes (Tarsons)
Slide
Needle (Dispovan, sterile needle, 0.45 x 13 mm)
Brush (Camel, Size 0)
Gas mask
Plant tissue (Leaf, root or whole seedling) collected freshly after stress treatment
Distilled water
Mercuric chloride (HgCl2) (SRL Sisco Research Laboratories, catalog number: 25699 )
Schiff’s reagents (HiMedia Laboratories, catalog number: S074-500ML )
Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) (HiMedia Laboratories, catalog number: GRM3903-500G )
Potassium nitrate (KNO3) (HiMedia Laboratories, catalog number: GRM1401-500G )
Potassium phosphate monobasic (KH2PO4) (HiMedia Laboratories, catalog number: RM3943-500G )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (HiMedia Laboratories, catalog number: RM684-5KG )
Boric acid (H3BO3) (HiMedia Laboratories, catalog number: MB007-1KG )
Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (HiMedia Laboratories, catalog number: RM685-500G )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (HiMedia Laboratories, catalog number: PCT0118-1KG )
Sodium molybdate dihydrate (Na2MoO4·2H2O) (HiMedia Laboratories, catalog number: GRM415-100G )
Copper(II) sulfate pentahydrate (CuSO4·5H2O) (HiMedia Laboratories, catalog number: RM630-500G )
Ferric chloride (FeCl3) (HiMedia Laboratories, catalog number: RM1178-1KG )
EDTA (HiMedia Laboratories, catalog number: RM678-100G )
Ammonium sulfate ((NH4)2SO4) (HiMedia Laboratories, catalog number: MB004-250G )
Potassium sulfate (K2SO4) (HiMedia Laboratories, catalog number: GRM404-500G )
Manganese sulfate monohydrate (MnSO4·H2O) (SRL Sisco Research Laboratories, catalog number: 1347151 , 500G)
Sodium Meta-bisulphate (Na2S2O5) (Sigma-Aldrich, catalog number: 255556-100G )
Calcium chloride dihydrate (CaCl2·2H2O) (HiMedia Laboratories, catalog number: PCT0004-500G )
Aluminum chloride (AlCl3) (Merck, catalog number: 8010810100 )
Cadmium chloride (CdCl2) (Thermo Fisher Scientific, catalog number: Q17584 )
Zinc chloride (ZnCl2) (Thermo Fisher Scientific, catalog number: Q28785 )
Ethanol (Diluent for DNA Extraction, HiMedia Laboratories, catalog number: MB228-500ML )
Glycerol (Sigma-Aldrich, catalog number: V800196-500ML )
Acetic acid (Thermo Fisher Scientific, catalog number: Q21057 )
Hydrochloric acid (HCl) (HiMedia Laboratories, catalog number: AS004-2.5L )
Schiff’s reagent staining solution (see Recipe 1)
Hoagland solution (see Recipe 2)
Modified nutrient solution (see Recipe 3)
Treatment solutions (AlCl3, CdCl2, ZnCl2) (see Recipe 4)
Sulfite solution (see Recipe 5)
Bleaching solution (see Recipe 6)
Storage solution (see Recipe 7)
Equipment
Pipettes (Gilson, PIPETMAN, 2-20,20-200 and 100-1,000 µl)
pH meter (pH Tutor, Eutech Instruments)
Magnetic stirrer cum hot plate (Tarsons)
Weighing balance (Sartorius, 0.1 mg-220 g)
Water bath (Equitron unstirred water bath, Medica Instruments)
Light microscope (Olympus, 10x objective)
Digital camera (Nikon, model: COOLPIX P100 , 10.3 megapixel, 26x zoom)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Awasthi, J. P., Saha, B., Chowardhara, B., Devi, S. S., Borgohain, P. and Panda, S. K. (2018). Qualitative Analysis of Lipid Peroxidation in Plants under Multiple Stress Through Schiff’s Reagent: A Histochemical Approach. Bio-protocol 8(8): e2807. DOI: 10.21769/BioProtoc.2807.
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Category
Plant Science > Plant biochemistry > Lipid
Biochemistry > Lipid > Membrane lipid
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2,808 | https://bio-protocol.org/exchange/protocoldetail?id=2808&type=0 | # Bio-Protocol Content
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Peer-reviewed
Extraction of Small Molecules from Fecal Samples and Testing of Their Activity on Microbial Physiology
EA Eduardo S. Alves
RF Rosana B. R. Ferreira
L. Caetano M. Antunes
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2808 Views: 6732
Edited by: David Cisneros
Reviewed by: Parul Mehrotra
Original Research Article:
The authors used this protocol in Jul 2017
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Jul 2017
Abstract
The human body is colonized by vast communities of microbes, collectively known as microbiota, or microbiome. Although microbes colonize every surface of our bodies that is exposed to the external environment, the biggest collection of microbes colonizing humans and other mammals can be found in the gastrointestinal tract. Given the fact that the human gut is colonized by several hundred microbial species, our group hypothesized that the chemical diversity of this environment should be significant, and that many of the molecules present in that environment would have important signaling roles. Therefore, we devised a protocol to extract these molecules from human feces and test their signaling properties. Potentially bioactive extracts can be tested through addition to culture medium and analyses of bacterial growth and gene expression, among other properties. The protocol described herein provides an easy and rapid method for the extraction and testing of metabolites from fecal samples using Salmonella enterica as a model organism. This protocol can also be adapted to the extraction of small molecules from other matrices, such as cultured mammalian cells, tissues, body fluids, and axenic microbial cultures, and the resulting extracts can be tested against various microbial species.
Keywords: Metabolome Microbiome Gut Small molecules Extraction Microbial signaling
Background
Complex assemblages of microbes live in and on humans, colonizing every surface exposed to the external environment. These communities have received several denominations over the decades, including normal flora, microbiota, and, more recently, microbiome (Sekirov et al., 2010; Kashyap et al., 2017). In humans, these vast microbial communities colonize our skin, respiratory tract, genitals, gastrointestinal tract, and many other sites. By far, the most heavily colonized site is the gastrointestinal tract, where trillions of microbes, encompassing several hundred species, coexist peacefully with their hosts. Some of these species knowingly live in symbiotic associations with the human organism, where both parts benefit from the interactions. For others, the relationship may be purely commensal, where the parts coexist without causing any harm to each other, but without providing or obtaining any benefit (Sekirov et al., 2010; Kundu et al., 2017).
The human gastrointestinal microbiota presents significant diversity in their composition, and stands out as a complex environment where interactions between different microbes as well as between microbes and host cells constantly occur. Bacteria are known to produce a plethora of bioactive small molecules, such as antibiotics, bacteriocins, pigments, secondary metabolites, quorum sensing signals, and many others (Antunes and Ferreira, 2009; Antunes et al., 2010; Antunes et al., 2011a). In such a complex environment as the intestinal microbiome, it is almost imperative to consider the production and accumulation of such molecules. These small molecules may represent by-products of metabolic activities or signals with specific roles, and can be produced both by the host itself as well as the microbes living in that environment; in many cases these small molecules are the tools used by these organisms to interact. Using high-throughput mass spectrometry-based metabolomics, we have previously shown that thousands of small molecules can be found in the lumen of the mammalian intestinal tract, and that the gut microbiome is involved in the production of many of them (Antunes et al., 2011b). In order to ascertain the signaling potential of small molecules from the gut metabolome, we have also extracted these molecules and tested their ability to modulate growth and gene expression of an enteric pathogen. As shown by our previous results, Salmonella enterica serovar Typhimurium responds to these molecules, and modulates the expression of over one hundred genes in response to bioactive small molecules from human feces (Antunes et al., 2014). Interestingly, many of the genes regulated by the fecal extract are required for the pathogenesis of Salmonella, such as those involved in the invasion of non-phagocytic host cells. More recently, we were able to purify and identify small aromatic compounds as the culprits for the regulation of Salmonella genes by the human gut metabolome (Peixoto et al., 2017). Here, we describe in detail the methods used by our group to obtain small molecules from the human gut metabolome and test them against Salmonella for various biological activities. A workflow of the procedures described herein can be found in Figure 1.
Figure 1. Workflow of the procedures described in this protocol
Materials and Reagents
Polypropylene container (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 193A )
Aluminum foil
Tape
Graduated glass pipettes (Fisher Scientific, catalog number: 13-678-25E )
2-ml Safe-Lock tubes (Eppendorf, catalog number: 0030120094 )
Syringes (Descarpack, catalog number: 0324501 )
Conical tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 362694 )
Axygen universal pipette tips (Corning, Axygen®, catalog number: T-200-C-L-R )
Barrier tips (Fisher Scientific, catalog number: 02-707-430 )
Syringe filter, 0.22-µm pore (KASVI, catalog number: K18-230 )
Cuvettes (BRAND, catalog number: 759115 )
Inoculation loop
Borosilicate tubes, 16 x 100 mm (DWK Life Sciences, Kimble, catalog number: 73500-16100 )
Bacterial culture (Salmonella enterica serovar Typhimurium SL1344)
HPLC-grade ethyl acetate, ≥ 99.7% pure (Sigma-Aldrich, catalog number: 34858 )
Nitrogen gas
HPLC-grade methanol (Sigma-Aldrich, catalog number: 34860 )
C18 cartridges containing 360 mg of sorbent (WATERS, catalog number: WAT051910 )
Distilled water
Phosphate-buffered saline (Sigma-Aldrich, catalog number: P5493-1L )
RNeasy Mini Kit (QIAGEN, catalog number: 74106 )
Agarose (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17850 )
QuantiTect Reverse Transcription Kit (QIAGEN, catalog number: 205311 )
MinElute Reaction Cleanup Kit (QIAGEN, catalog number: 28204 )
Primers (Integrated DNA Technologies)
Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4367659 )
Hydrochloric acid, 36.5-38% (Sigma-Aldrich, catalog number: H1758 )
Sodium hydroxide, ≥ 98% pure (Sigma-Aldrich, catalog number: S8045 )
Luria-Bertani (LB) Broth (BD, catalog number: 244620 )
Bacteriological agar (Sigma-Aldrich, catalog number: A5306 )
Streptomycin (Sigma-Aldrich, catalog number: S9137-25G )
1 N HCl (see Recipes)
1 N NaOH (see Recipes)
LB broth or agar (see Recipes)
LB with streptomycin (see Recipes)
Equipment
Digital scale (Ohaus, model: SP202 )
Glass bottle (Fisher Scientific, catalog number: FB800500 )
Orbital shaker (BiomiXer, model: TS-2000A )
Graduated glass cylinder (Laborglas, catalog number: 91376 )
Glass boiling flask (Corning, PYREX®, catalog number: 4100-125 )
Electronic pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 9501 )
Fume hood
Rotary evaporator (Heidolph, catalog number: 560-01300-00 )
37 °C incubator
Freezer, -20 °C
P20 micropipette (Gilson, catalog number: F123600 )
P200 micropipette (Gilson, catalog number: F123601 )
P1000 micropipette (Gilson, catalog number: F123602 )
Speed-Vac concentrator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SPD131DDA-115 )
Vortex (Scientific Industries, model: Vortex-Genie 2, catalog number: SI-0236 )
pH meter (Sigma-Aldrich, catalog number: MT 51302916 )
Manufacturer: Mettler-Toledo, catalog number: 51302916 .
Autoclave
Visible range spectrophotometer
Nucleic acid (UV) spectrophotometer
Shaker (NOVATECNICA, model number: NT145 )
Centrifuge for 96-well plates (Eppendorf, model: 5810 , catalog number: 5810000017)
Real Time PCR Machine (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4376357 )
Software
GraphPad Prism (GraphPad Software)
StepOne Software v2.3 (Thermo Fisher Scientific)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Alves, E. S., Ferreira, R. B. R. and Antunes, L. C. M. (2018). Extraction of Small Molecules from Fecal Samples and Testing of Their Activity on Microbial Physiology. Bio-protocol 8(8): e2808. DOI: 10.21769/BioProtoc.2808.
Download Citation in RIS Format
Category
Microbiology > Microbial signaling > Quorum sensing
Biochemistry > Other compound > Small molecular
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2,809 | https://bio-protocol.org/exchange/protocoldetail?id=2809&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Quantifying Gene Expression Directly from FACS Using Hydrolysis (TaqMan) Probes on Flash-frozen Cells
Kirsten A. Copren
OT Oyang Teng
CA Cassandra J. Adams
Cláudia Bispo
ML Michael Lee
Published: Apr 20, 2018
DOI: 10.21769/BioProtoc.2809 Views: 8167
Edited by: Jia Li
Reviewed by: Alexandros AlexandratosPrashanth N Suravajhala
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Abstract
The TaqMan Gene Expression Cells-to-CtTM Kit enables reverse transcription directly from cell lysates without the need for isolating RNA. The recommended input range for the lysis reaction is between 10-105 cells, though the upper limit may vary somewhat according to cell type. This protocol is partially adapted from the manufacturer’s protocol (TaqMan Gene Expression Cells-to-CtTM Kit User Guide) for gene expression for use with frozen cell pellets to allow for sample transfer among laboratories and involves experimentally confirmed dilution prior to lysis. Within this constraint, the recommended range for sample cell number for frozen cells is between 40-2 x 105 cells. This protocol is also designed for use with a ‘homebrew’ qPCR master mix as the volume of TaqMan Genotyping Master Mix included in the kit may be insufficient for analysis of large numbers of samples and/or assays (Smythe and Copren, 2008). Cell count is provided by fluorescence-activated cell sorting (FACS) and is a unique aspect of this protocol.
Keywords: Quantitative PCR (qPCR) Reverse transcription Cell lysis Flow cytometry Cq values Cell pellets Cell sorting FACS Gene expression TaqMan assays Hydrolysis probes
Background
This protocol is a modification and experimental validation of the TaqMan Gene Expression Cells-to-CtTM Kit User Guide, Publication Number 4385117 Revision E (2012) (Thermo Fisher Scientific/Life Technologies) for flash-frozen, rather than fresh, cells to allow for transportation among core facility laboratories (Procedure B). Using fresh cells requires all steps of the process to be performed sequentially with no stopping point at one facility. Using flash-frozen cells allows the separation of the cell sorting and downstream steps at different locations while ensuring the viability of the cells. The cell sorting is unique to this protocol. It is important to maintain the viability of the cells to ensure the accurate measurement of cellular genomes and transcriptomes (Gawad et al., 2016). Flash-freezing also provides a stopping point for sorting the cells and the downstream analysis. This protocol is also designed and validated for use with a ‘homebrew’ qPCR master mix allowing the analysis of gene expression from a larger number of samples and/or assays than originally allocated by the manufacture’s kit.
We attempt to consistently use the nomenclature recommended by the MIQE guidelines for qPCR (Bustin et al., 2009). Thermo Fisher Scientific uses Cts (cycles at threshold), and their kit is named as such, but the recommended standardization is Cqs (quantification cycles). ‘TaqMan’ probes/assays are standardized as hydrolysis probes.
Materials and Reagents
Pipette tips
10 ml sterile pipette tip
25 ml sterile pipette tip
1.5 or 1.7, 5, 10, 50 ml tubes (USA Scientific, VWR)
0.2 ml strip tubes (USA Scientific)
384 well optical plate (MicroAmpTM Optical 384, Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4343370 )
Liquid nitrogen
TaqMan Gene Expression Cells-to-CtTM Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: 4399002 ):
Lysis Solution (P/N 4383583)
DNase I (P/N 4386321)
Stop Solution (P/N 4386318)
20x Reverse Transcriptase (P/N 4386319)
2x Reverse Transcription Buffer (P/N 4383586)
Nuclease-free water (Sigma-Aldrich)
GAC 5x QPCR Buffer without Taq polymerase (see Recipes)
GeneAmpTM 10x PCR Buffer II (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4379878 )
Glycerol (Sigma-Aldrich)
Gelatin (Sigma-Aldrich)
Deionized water (Sigma-Aldrich)
ROX Passive Reference Dye (IDTDNA)
Taqman Gene Expression Assay: Hs03003631_g1 (Eukaryotic 18S RNA) (Thermo Fisher Scientific)
Universal cDNA (various vendors or homemade)
Equipment
Pipettes
Small volume pipettor
Large volume pipettor
Cell sorter: FACSAria II (BD, model: FACSAria II )
Centrifuge
Thermal cycler (Bio-Rad Laboratories, model: C1000 TouchTM )
7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: 7900HT )
-80 °C freezer
Procedure
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Category
Molecular Biology > RNA > qRT-PCR
Cancer Biology > General technique > Molecular biology technique
Cell Biology > Cell-based analysis > Flow cytometry
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281 | https://bio-protocol.org/exchange/protocoldetail?id=281&type=0 | # Bio-Protocol Content
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Isolation of Human Blood Progenitor and Stem Cells from Peripheral Blood by Magnetic Bead
Salma Hasan
Isabelle Plo
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.281 Views: 15837
Original Research Article:
The authors used this protocol in Mar 2012
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Mar 2012
Abstract
The antigen CD34 is a well-known marker present on human progenitor and stem cells. This protocol explains the isolation of CD34+ cells from peripheral blood using magnetic bead separation technique. The approximate abundance of CD34+ cells in blood is 0.1% of mononuclear cells.
Keywords: Hematopoiesis Progenitors Isolation
Materials and Reagents
CD34+ cells
Peripheral blood sample (at least 50 ml)
Dextran solution (Sigma-Aldrich, catalog number: D1037-500G )
PBS (Life Technologies, Invitrogen™, catalog number: 14190-094 )
Ficoll human (PAA Laboratories GmbH, catalog number: P04-60500 )
Fetal calf serum (FCS) (Hyclone, catalog number: SV30160.03 )
CD34 MicroBeads and FcR blocking reagent (Miltenyi Biotec, catalog number: 130-046-702 )
APC mouse anti-human CD34 antibody (BD Biosciences, catalog number: 555824 )
EDTA
2% dextran solution
1 L 2% dextran solution (see Recipes)
Equipment
Centrifuges
Auto MACS Pro separator (Miltenyi Biotec)
30 μm nylon mesh (Miltenyi Biotec, catalog number: 130-041-407 )
Tissue culture hood
Flow cytometer
Vacuum filter unit (22 μm, GP Millipore Express PLUS membrane)
Procedure
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Category
Stem Cell > Adult stem cell > Hematopoietic stem cell
Cell Biology > Cell isolation and culture > Cell isolation
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2,810 | https://bio-protocol.org/exchange/protocoldetail?id=2810&type=0 | # Bio-Protocol Content
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In-vitro and in-planta Botrytis cinerea Inoculation Assays for Tomato
JL Jiajie Lian*
HH Hongyu Han*
JZ Jiuhai Zhao
Chuanyou Li
*Contributed equally to this work
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2810 Views: 12622
Reviewed by: Tuan Minh Tran
Original Research Article:
The authors used this protocol in Aug 2017
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Abstract
Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key procedures to investigate the mechanism of plant immunity. Here we describe a protocol for B. cinerea inoculation on medium and planta based on our study using the tomato-B. cinerea system.
Keywords: Botrytis cinerea Tomato Inoculation
Background
B. cinerea causes serious loss in more than 200 crops worldwide, including many important vegetables and small fruit crops. The broad-spectrum pathogen can infect plant stem, leaf, flower and fruit to produce spores (Dean et al., 2012; van Kan et al., 2017), which prefer to occur under high humidity (Elad et al., 2007). The produced spores pose long lasting threat to diverse hosts (Elad et al., 2007). Based on its scientific and economic importance, B. cinerea was ranked as the second most important plant-pathogenic fungus (Dean et al., 2012). Among B. cinerea host plants, tomato (Solanum lycopersicum), an economically valuable species, also serves as a classic model to study plant immunity (Ryan, 2000; Sun et al., 2011; Rosli and Martin, 2015). To investigate the molecular basis of plant immunity to B. cinerea, we employ a routine procedure to produce B. cinerea spores on artificial media. In addition, we provide detailed methods to infect tomato plants or detached leaves with a controlled strength using the collected spores and quantify disease development. This protocol has been successfully used to reveal the transcriptional regulation of master regulator MYC2 in Jasmonate-mediated plant immunity (Du et al., 2017).
Materials and Reagents
General lab materials, including:
Nylon mesh (Solarbio, catalog number: YA0964 )
Petri dish (9 cm)
Square Petri dish (10 x 10 cm)
Funnel (Conventional type, upper diameter: 8 cm)
1.5 ml microcentrifuge tube (USA Scientific, catalog number: 4036-3204)
Manufacturer: Eppendorf, catalog number: 022363204 .
15 ml centrifuge tube (Corning, catalog number: 430790 )
50 ml centrifuge tube (Shanghai Kirgen, catalog number: KG2821 )
Pipette tips
Micropore tape (3M, MicroporeTM, catalog number: 1530S-1 )
Nutritional soil (Miracle-Gro® Garden Soil for Vegetables with total N 0.68%, P2O5 0.27% and K2O 0.36%)
Tomato (Solanum lycopersicum) cv M82
B. cinerea B05.10
V8 juice (Campbell, 100%, original vegetable juice)
Calcium carbonate (CaCO3) (Sigma-Aldrich, catalog number: V900138 )
Bacto-agar (BD, BactoTM, catalog number: 214010 )
Mycological peptone (Sigma-Aldrich, catalog number: 77199 )
Sodium phosphate (Sigma-Aldrich, catalog number: 342483 )
Maltose (Sigma-Aldrich, catalog number: M5885 )
2x V8 agar medium (see Recipes)
SMB medium (see Recipes)
0.8% agar medium (see Recipes)
Equipment
Pipettes (Gilson, Pipetman® G)
Water bath (YIHENG, model: DK-80 )
Autoclave (Panasonic Healthcare, model: MLS-3781L )
Customized transparent plastic box (materials: Polymeric Methyl Methacrylate, L/W/H: 50/50/50 centimeter, open at the top, Figure 1A) or any incubators that can be used alternatively
Centrifuge (Eppendorf, models: 5810 R and 5424 )
Microscope (ZEISS, model: Axio Imager Z2 ; 20x objective plan-APOCHROMAT; 10x objective EC plan-NEOFLUAR) or other light microscopes
Counting chamber (QIUJING® KB-K-25, 0.1 mm, 1/400 mm2)
Software
ImageJ (https://imagej.nih.gov/ij/download.html)
Microsoft Excel
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lian, J., Han, H., Zhao, J. and Li, C. (2018). In-vitro and in-planta Botrytis cinerea Inoculation Assays for Tomato. Bio-protocol 8(8): e2810. DOI: 10.21769/BioProtoc.2810.
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Category
Plant Science > Plant immunity > Disease bioassay
Microbiology > Microbe-host interactions > In vitro model
Microbiology > Microbe-host interactions > Virus
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2,811 | https://bio-protocol.org/exchange/protocoldetail?id=2811&type=0 | # Bio-Protocol Content
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Testing Effects of Chronic Chemogenetic Neuronal Stimulation on Energy Balance by Indirect Calorimetry
Sangho Yu
HM Heike Münzberg
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2811 Views: 5110
Edited by: Oneil G. Bhalala
Reviewed by: Saswata Sankar Sarkar
Original Research Article:
The authors used this protocol in May 2016
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Abstract
The fundamental of neuroscience is to connect the firing of neurons to physiological and behavioral outcomes. Chemogenetics enables researchers to control the activity of a genetically defined population of neurons in vivo through the expression of designer receptor exclusively activated by designer drug (DREADD) in specific neurons and the administration of its synthetic ligand clozapine N-oxide (CNO) (Sternson and Roth, 2014). Using stimulatory Gq-coupled DREADD (hM3Dq) in mice, we showed that leptin receptor (LepRb)-expressing neurons in the preoptic area (POA) of the hypothalamus are warm-sensitive neurons that mediate warm-responsive metabolic and behavioral adaptations by reducing energy expenditure and food intake (Yu et al., 2016). We also used DREADD technology to test effects of chronic stimulation of POA LepRb neurons on energy expenditure, food intake, and body weight with the TSE indirect calorimetry system. Here we describe the detailed protocol of how we used indirect calorimetry to study the outcome of chronic stimulation of POA LepRb neurons. This protocol can be adapted to study long-term metabolic and behavioral consequences of other neuronal modulations, with possible modifications to the type of DREADD, duration of CNO treatment, or method of CNO delivery.
Keywords: Chemogenetics DREADD Energy expenditure Food intake Indirect calorimetry TSE
Background
The POA is a central hub for body temperature homeostasis, which receives thermosensory information from the periphery and tunes the degree of sympathetic output to brown adipose tissue (BAT), cutaneous blood vessels, and heart to control the amount of heat generation and dissipation (Nakamura, 2011). We discovered that LepRb neurons in the POA are stimulated by warm ambient temperature and mediate warm-adaptive responses that include suppression of BAT thermogenesis and food intake (Yu et al., 2016). This discovery was made mainly through chemogenetic stimulation of POA LepRb neurons by virally expressing Gq-coupled DREADD, hM3Dq, in POA LepRb neurons and injecting CNO in mice. A single IP injection of CNO can stimulate target neurons up to 10 h (Krashes et al., 2011; Rezai-Zadeh et al., 2014). This long-lasting CNO effect allows researchers to modulate target neuron activity chronically with two IP injections of CNO per day.
Optogenetics and chemogenetics have revolutionized the field of neuroscience by providing tools to selectively manipulate target neuron activity with its own unique advantages and disadvantages. For studying neurons that modulate energy balance, researchers often use an indirect calorimetry system to simultaneously measure energy expenditure and food intake for an extended period of time (Rezai-Zadeh et al., 2014; Correa et al., 2015; Qualls-Creekmore et al., 2017). Chemogenetics is best suited for this type of study thanks to slow clearance of CNO from the body and no requirement of optical devices as in optogenetics. In our study, we investigated consequences of chronic stimulation of POA LepRb neurons by DREADD with the PhenoMaster indirect calorimetry system. CNO was injected at 0.3 mg/kg twice per day for six days, and energy expenditure and food intake were continuously measured every 25 min. Body weight was measured once every morning to monitor how changed energy expenditure and food intake affected body weight. In this protocol, we describe a step-by-step procedure of using the TSE PhenoMaster indirect calorimetry system to measure energy expenditure and food intake during chronic stimulation of POA LepRb neurons by DREADD in mice.
Materials and Reagents
½ CC Lo-dose U-100 insulin syringe 28 G ½ (BD, catalog number: 329461 )
1.5 ml microcentrifuge tube (SARSTEDT, catalog number: 72.690.301 )
LepRb-Cre mice
Notes:
In-house bred and derived from original breeders kindly provided by Dr. Martin Myers, Jr., University of Michigan (Leshan et al., 2006); that were injected with AAV5-hSyn-DIO-mCherry (Vector Core, University of North Carolina at Chapel Hill) in the POA (control group, 3 month old, 3 males and 2 females, n = 5).
LepRb-Cre mice that were injected with AAV5-hSyn-DIO-hM3Dq-mCherry (Vector Core, University of North Carolina at Chapel Hill, kindly made available by Dr. Bryan Roth) in the POA (experimental group, 3 month old, 3 males and 2 females, n = 5).
All experiments were approved by the Institutional Animal Care and Use Committee at Pennington Biomedical Research Center.
CNO (Sigma-Aldrich, catalog number: C0832 )
Saline/0.9% NaCl (B. Braun Medical, catalog number: L8001 )
CNO stock (see Recipes)
CNO working solution (see Recipes)
Equipment
PhenoMaster (TSE systems)
Scale (Ohaus, Scout-Pro, model: SPE601 )
Vortex Mixer (American Scientific Products, catalog number: S8223-1 )
Software
LabMaster V5.0.8 (TSE systems)
Excel 2010 (Microsoft)
SPSS 22 (IBM)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Yu, S. and Munzberg, H. (2018). Testing Effects of Chronic Chemogenetic Neuronal Stimulation on Energy Balance by Indirect Calorimetry. Bio-protocol 8(8): e2811. DOI: 10.21769/BioProtoc.2811.
Yu, S., Qualls-Creekmore, E., Rezai-Zadeh, K., Jiang, Y., Berthoud, H. R., Morrison, C. D., Derbenev, A. V., Zsombok, A. and Munzberg, H. (2016). Glutamatergic preoptic area neurons that express leptin receptors drive temperature-dependent body weight homeostasis. J Neurosci 36(18): 5034-5046.
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Category
Neuroscience > Behavioral neuroscience > Animal model
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2,812 | https://bio-protocol.org/exchange/protocoldetail?id=2812&type=0 | # Bio-Protocol Content
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Determination of Intracellular Osmolytes in Cyanobacterial Cells
XT Xiaoming Tan
KS Kuo Song
CQ Cuncun Qiao
XL Xuefeng Lu
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2812 Views: 5215
Reviewed by: Valentine V Trotter
Original Research Article:
The authors used this protocol in Jun 2017
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Abstract
Most of the cyanobacteria accumulate osmolytes including sucrose, glucosylglycerol, in their cells in response to salt stress. Here we describe a protocol of our laboratory for extraction and quantification of cyanobacterial intracellular sucrose and glucosylglycerol. We have confirmed this protocol was applicable to at least four kinds of cyanobacteria, filamentous cyanobacterium Anabaena sp. PCC 7120, unicellular cyanobacterium Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942 and halotolerant unicellular cyanobacterium Synechococcus sp. PCC 7002.
Keywords: Osmolyte Sucrose Glucosylglycerol Synechocystis Cyanobacteria Ion chromatography
Background
Osmolytes (or compatible solutes) are a group of low-molecular-weight organic solutes, and play important physiological roles on abiotic stress acclimation in microbes including cyanobacteria (Reed and Stewart, 1985; Klähn and Hagemann, 2011; Slama et al., 2015). For determination of intracellular osmolytes from cyanobacterial cells, several protocols have been established (Reed and Stewart, 1985; Hagemann et al., 1997; Motta et al., 2004; Du et al., 2013; Fa et al., 2015).
Among these methods, nuclear magnetic resonance (NMR) spectroscopy based method was the only one which could be directly applied on microbe cultures without any extraction procedure (Motta et al., 2004). However, this protocol has just been tested in cultures of Halomonas pantelleriensis and Sulfolobus solfataricus rather than in cyanobacterial cultures. For all other methods, 80% ethanol was used for extraction of osmolytes from cyanobacterial cells. After derivatization by some trimethyl-silyl reagents, the derivatives of osmolytes could be analyzed by gas chromatography (Reed and Stewart, 1985). Alternatively, the extracted osmolytes could be directly analyzed by high-performance liquid chromatography (HPLC) (Hagemann et al., 1997). Our lab has firstly reported our protocol for osmolyte determination by ion chromatography (IC) equipped with a carb-Pac®MA1 analytical column (Du et al., 2013). In this protocol (Du et al., 2013), the column was equilibrated with 600 mM NaOH with a flow rate of 0.4 ml/min, and the running time for one sample was 45 min. Later, the concentration of NaOH was increased to 800 mM, and the running time for each sample was shortened to 32 min (Song et al., 2016 and 2017). Recently, the PA10 analytical column was successfully used for osmolyte analysis by ion chromatography (Qiao et al., 2017), and the running time for one sample was further shortened to 10 min. It is worthy to note that our collaborator has established a novel method for osmolyte determination by combination of separation by capillary ion chromatography and detection by mass spectrometry (CIC-MS) (Fa et al., 2015). The NMR and CIC-MS based methods are suitable for determination of unknown osmolytes from cyanobacteria. Compared with these two methods as well as the GC and HPLC based methods, our IC based method has advantages on running time and accuracy which would be helpful for the high throughput osmolyte detection used in some cyanobacterial metabolic engineering reseaches.
Therefore, we detailedly present our recent IC-based protocol here for osmolyte determination in cyanobacterial cells.
Materials and Reagents
2 ml microcentrifuge tubes (Cypress
10 ml Centrifuge tube, Snap-Cap (Kangjian)
1 ml syringe (Jianshi)
Syringe membrane filters, 0.22 μm (Jinteng)
Synechocystis sp. PCC 6803
Nitrogen (Dehai)
CO2 (Dehai)
MilliQ water (Millipore, Germany)
Potassium phosphate dibasic trihydrate (K2HPO4∙3H2O) (Sinopharm Chemical Reagent, catalog number: 10017518 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10013018 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sinopharm Chemical Reagent, catalog number: 20011160 )
Critic acid (Sinopharm Chemical Reagent, catalog number: 10007118 )
Ferric ammonium citrate (Sinopharm Chemical Reagent, catalog number: 30011428 )
EDTA·2Na (Sinopharm Chemical Reagent, catalog number: 10009717 )
Sodium carbonate (Na2CO3) (Sinopharm Chemical Reagent, catalog number: 10019260 )
Boric acid (H3BO3) (Sinopharm Chemical Reagent, catalog number: 10004818 )
Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sinopharm Chemical Reagent, catalog number: 20026118 )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10024018 )
Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sinopharm Chemical Reagent, catalog number: 10019818 )
Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sinopharm Chemical Reagent, catalog number: 10008218 )
Cobalt(II) chloride hexahydrate (CoCl2·6H2O) (Sinopharm Chemical Reagent, catalog number: 10007216 )
Sodium nitrate (NaNO3) (Sinopharm Chemical Reagent, catalog number: 10019918 )
Sodium chloride (NaCl) (Sinopharm Chemical Reagent, catalog number: 10019318 )
Ethanol (Sinopharm Chemical Reagent, catalog number: 10009218 )
Glycerol standard (99%, Sinopharm Chemical Reagent, catalog number: 10010618 )
Glucosylglycerol standard (50%, Bitop, http://www.bitop.de/en/products/cosmetic-active-ingredients/glycoin)
Glucose standard (Sinopharm Chemical Reagent, catalog number: 10010518 )
Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
50% Sodium chloride (NaOH) solution (Thermo Fisher Scientific)
BG11 medium (see Recipes)
Saturated NaCl solution prepared in BG11 media (see Recipes)
80% ethanol (see Recipes)
Osmolytes standards (see Recipes)
200 mM NaOH (see Recipes)
Equipment
200, 1,000 ml Pipettes (Eppendorf, Germany)
Glass column photobioreactors (Sanhehuaxing, China) (Tan et al., 2011)
Centrifuge (Sigma-Zentrifugen, model: Sigma 1-14 )
Water bath (Yarong, model: B260 )
Organomation (Hengao, model: HGC-24A )
Ion chromatography (Thermo Fisher Scientific, Thermo ScientificTM, model: DionexTM ICS-5000+ )
DinexTM CarboPacTM analytical column (4 x 250 mm, Thermo Fisher Scientific, model: DinexTM CarboPacTM PA10 )
-80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: Forma 705 )
Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
Autoclave (Hirayama, model: HV-50 )
Software
Chromeleon software (version 6.80, Thermo Fisher Scientific)
IBM SPSS Statistics (IBM, version 19)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Tan, X., Song, K., Qiao, C. and Lu, X. (2018). Determination of Intracellular Osmolytes in Cyanobacterial Cells. Bio-protocol 8(8): e2812. DOI: 10.21769/BioProtoc.2812.
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Category
Microbiology > Microbial metabolism > Carbohydrate
Microbiology > Microbial physiology > Adaptation
Biochemistry > Carbohydrate > Polysaccharide
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2,813 | https://bio-protocol.org/exchange/protocoldetail?id=2813&type=0 | # Bio-Protocol Content
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Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System
KL Kuancheng Liu
JH Jianming Hu
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2813 Views: 5756
Edited by: Yannick Debing
Original Research Article:
The authors used this protocol in Jun 2016
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Abstract
The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions. We have recently developed a mammalian cell-free system based on the rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological concentrations and assembles into capsids under near-physiological conditions. This system has already revealed HBc assembly requirements that are not anticipated based on previous assembly systems. Furthermore, capsid assembly in this system is regulated by endogenous host factors that can be readily manipulated. Here we present a detailed protocol for this cell-free capsid assembly system, including an illustration on how to manipulate host factors that regulate assembly.
Keywords: Hepatitis B virus Cell-free capsid assembly system Rabbit reticulocyte lysate Phosphorylation RNA binding
Background
The hepatitis B virus (HBV) is an important global human pathogen that chronically infects hundreds of millions of people worldwide and represents a major cause of viral hepatitis, liver cirrhosis, and liver cancer (Seeger et al., 2013; Trepo et al., 2014). HBV replicates its genomic DNA, a relaxed circular, partially duplex DNA (RC DNA), via reverse transcription of an RNA intermediate, the so-called pregenomic RNA (pgRNA), within a nucleocapsid (NC) (Summers and Mason, 1982; Hu and Seeger, 2015; Hu, 2016), which packages a copy of pgRNA together with the virally encoded reverse transcriptase (RT) protein (Bartenschlager and Schaller, 1992; Hu and Lin, 2009). It is within NCs that the RT converts pgRNA into RC DNA.
The icosahedral HBV capsid shell consists of multiple copies of a single viral protein, the HBV core (capsid) protein (HBc). HBc is composed of an N-terminal domain (NTD, aa 1-140) and a C-terminal domain (CTD, 150-183 or 185 depending on strains), which are connected by a linker region (141-149). In heterologous overexpression systems including bacteria and insect cells and in vitro assembly reactions using high concentrations of purified HBc and/or salt, NTD alone, without CTD, is sufficient for capsid assembly; thus it is also called the assembly domain (Gallina et al., 1989; Birnbaum and Nassal, 1990; Lanford and Notvall, 1990; Wingfield et al., 1995). Though not required for capsid assembly in these systems, the highly basic and arginine-rich CTD shows non-specific nucleic acid binding activities (Hatton et al., 1992) and plays important roles in viral RNA packaging (Nassal, 1992), DNA synthesis (Nassal, 1992; Yu and Summers, 1994), and nuclear import of capsids (Liao and Ou, 1995; Liu et al., 2015), all of which is further regulated by the dynamic phosphorylation state of CTD controlled by host factors (Kann and Gerlich, 1994; Kann et al., 1999; Daub et al., 2002; Ludgate et al., 2012; Liu et al., 2015). Whether CTD, and its state of phosphorylation, play a role in capsid assembly under physiological conditions remained unclear.
To study HBV capsid assembly under more physiological conditions, we have recently developed a mammalian cell-free assembly system based on the commonly used mammalian cell extract, rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological (low) concentrations (25-50 nM, monomer) and assembles into capsids under near-physiological conditions (Ludgate et al., 2016). This system allowed us to reveal an unexpected role of CTD in capsid assembly, which is further subjected to regulation by the state of CTD phosphorylation as controlled by endogenous host factors (Ludgate et al., 2016). This protocol is adapted from Ludgate et al. (2016) and more detailed information on this cell-free capsid assembly system is included, and different treatments are applied to address the roles of viral and host factors, such as RNA-binding activities of CTD and host phosphatases, in HBV capsid assembly under near-physiological conditions. This protocol will facilitate detailed studies on capsid assembly and host regulation under physiological conditions and identification of novel antiviral agents targeting HBc.
Materials and Reagents
Pipette tips (Denville Scientific, catalog numbers: P1096-FR , P1121 , P1122 , P1126 )
1.5 ml microcentrifuge tube (Denville Scientific, catalog number: C2170 )
Gloves and lab coat (Denville Scientific, catalog number: G4162 ; Medline Industries, catalog number: 83044QHW )
Proteinase K, RNA grade (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25530049 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509-1KG )
Phenol solution (Sigma-Aldrich, catalog number: P4557 )
Chloroform (Fisher Scientific, catalog number: BP1145-1 )
Sodium acetate (Sigma-Aldrich, catalog number: S2889-1KG )
Ethyl alcohol (EtOH) (AmericanBio, catalog number: AB00515-00100 )
UltraPureTM DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15510027 )
TNT® Coupled Rabbit Reticulocyte Lysate (RRL) (Promega, catalog number: L4610 )
EasyTagTM L-[35S]-Methionine (PerkinElmer, catalog number: NEG709A001MC )
RNasin® Plus Ribonuclease Inhibitor (Promega, catalog number: N2611 )
RNaseZapTM RNase Decontamination Solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
RNase A, DNase and protease-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0531 )
NEBuffer 3 (New England Biolabs, catalog number: B7003S )
Alkaline Phosphatase, Calf Intestinal (CIAP) (New England Biolabs, catalog number: M0290S )
Tris Base (Fisher Scientific, catalog number: BP152-10 )
Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: E5134 )
Sodium fluoride (Sigma-Aldrich, catalog number: S7920 )
Sodium pyrophosphate tetrabasic decahydrate (Sigma-Aldrich, catalog number: S6422 )
β-Glycerophosphate (Sigma-Aldrich, catalog number: G6251 )
Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 04693132001 )
TE buffer (see Recipes)
10x phosphatase inhibitors (PPI) (see Recipes)
25x protease inhibitor (see Recipes)
Equipment
Pipettes (Gilson, P1000, P200, P20, P2)
Fume hood (e.g., Protector Xstream Laboratory Hood, Labconco)
30 °C/37 °C Oven (SciGene, Robbins Scientific, model: Model 400 )
Microcentrifuge (Fisher Scientific, FisherbrandTM, model: accuSpinTM Micro 17 )
Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM1000 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Liu, K. and Hu, J. (2018). Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System. Bio-protocol 8(8): e2813. DOI: 10.21769/BioProtoc.2813.
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Category
Microbiology > Heterologous expression system > in viro translation
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Structure
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2,814 | https://bio-protocol.org/exchange/protocoldetail?id=2814&type=0 | # Bio-Protocol Content
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Peer-reviewed
Method for CRISPR/Cas9 Mutagenesis in Candida albicans
ND Neta Dean
HN Henry Ng
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2814 Views: 11153
Edited by: David Cisneros
Reviewed by: Emmanuel Orta-ZavalzaSneh Lata Singh
Original Research Article:
The authors used this protocol in Apr 2017
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Abstract
Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that allow simple ligation of synthetic duplex 23mer guide oligodeoxynucleotides for high copy gRNA expression in C. albicans strains that express codon-optimized Cas9. This protocol allows isolation and characterization of deletion strains within nine days.
Keywords: Candida albicans Cas9 CRISPR Fungal genetics gRNA Yeast
Background
C. albicans is a difficult organism to manipulate genetically. Since it normally exists as a diploid that does not readily undergo sexual reproduction, homozygous recessive mutations require sequential modification of each locus. The development and application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mutagenesis in C. albicans facilitates genetic manipulations because it allows simultaneous mutation of both alleles (Vyas et al., 2015; Min et al., 2016; Ng and Dean, 2017). CRISPR gene editing involves recruitment of an RNA-guided nuclease to a complementary target site adjacent to an NGG protospacer adjacent motif (PAM) (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013). CRISPR associated (Cas) nuclease is targeted with high specificity through complementary base pairing between a guide RNA associated with trans-activating CRISPR RNA (tracrRNA), which binds Cas9 (Gasiunas et al., 2012). Since the chromosomal target sequence is only ~20 nucleotides, expression of a single guide RNA (sgRNA) fused to a ~80 nucleotide tracrRNA, along with Cas9, is sufficient for targeted double-strand DNA cleavage. Since chromosomal breaks are lethal, there is a strong selective pressure for double stranded break repair. In C. albicans, co-expression of a donor repair fragment, containing homology to regions flanking the break, allows repair of the break by homologous recombination. Thus, appropriate design of the donor repair fragment allows introduction of sequence deletions, replacements or other chromosomal alterations.
Our previous studies demonstrated that a key factor contributing to high efficiency CRISPR mutagenesis of C. albicans genes relies on optimal gRNA expression (Ng and Dean, 2017). Toward this end, we created gRNA expression vectors that permit high levels of gRNA expression. The basis for this high-level expression is in part due to the presence of a strong, RNA polymerase II promoter (PADH1). This promoter drives the expression of an sgRNA flanked by a 5’ tRNA and a 3’ hepatitis delta virus (HDV) ribozyme RNA. The presence of these 5’ and 3’ flanking RNA sequences serve to promote efficient post-transcriptional processing to produce a mature sgRNA with precise ends. In the presence of an appropriate donor repair fragment, this increased sgRNA expression dramatically improves CRISPR/Cas mutagenesis in C. albicans. In practice, execution of mutagenesis is quite simple. The gRNA expression plasmid cloning relies on the annealing and ligation of two short gRNA encoding oligonucleotides into pre-cut vectors. Mutagenesis involves co-transformation of an sgRNA plasmid and the donor repair fragment into C. albicans strains that express codon-optimized Cas9 nuclease. Described below are detailed protocols for the design, synthesis, and cloning of gRNA oligonucleotides, healing fragment construction, yeast transformations, and mutant verification.
Materials and Reagents
1.5 ml microfuge tubes (acceptable as sold by any science distributor)
Glass or disposable round bottom sterile tubes for growing 2-5 ml cultures of yeast and bacteria (acceptable as sold by any science distributor)
Plastic Petri dishes (acceptable as sold by any science distributor)
PCR tubes
Toothpicks
Yeast strains (Table 1)
Table 1. Strain list
Plasmids (Table 2)
Table 2. Plasmids
E. coli competent cells (DH5α) (made as described, e.g., https://www.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells or can be purchased from New England Biolabs)
Note: We use CaCl2-treated DH5α but these can be purchased.
Glycerol (any source) (50% in H2O, autoclaved)
Salmon sperm DNA (Sigma-Aldrich, catalog number: D1626 ; 10 mg/ml, sonicated and boiled)
SapI (New England Biolabs, catalog number: R0569S )
ClaI (New England Biolabs, catalog number: R0197S )
Calf Intestinal Phosphatase (CIP) (New England Biolabs, catalog number: M0290L )
Custom oligonucleotides (between 18-60 nucleotides in length, Eurofins MWG Operon, Alabama)
T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
T4 ligase (New England Biolabs, catalog number: M0202L )
2x YT (see https://www.elabprotocols.com/protocols/#!protocol=5436 for recipe) (+ 100 µg/ml ampicillin) liquid media and plates (bacterial selection)
Ampicillin (any source)
Taq polymerase (any source)
Polyethylene glycol (PEG) 3350 (Sigma-Aldrich, catalog number: P4338 )
Lithium acetate (any source)
NcoI or StuI
Uridine (any source)
0.2% SDS
Agarose (any source)
Qiaquick Gel Extraction Kit (QIAGEN, catalog number: 28706 )
LB
Yeast extract (BD, BactoTM, catalog number: 212750 )
Tryptone (BD, BactoTM, catalog number: 211705 )
Sodium chloride (NaCl, certified ACS grade ≥ 99.0%) (Fisher Scientific, catalog number: S271 )
YPD
Yeast extract (BD, BactoTM, catalog number: 212750 )
Peptone (BD, BactoTM, catalog number: 211677 )
Dextrose (Fisher Scientific, catalog number: D16 )
Note: Make 20% stock in double distilled water.
Synthetic dextrose lacking uracil (SD-URA) liquid media and plates (uracil prototrophic yeast selection) (see http://cshprotocols.cshlp.org/content/2015/2/pdb.rec085639.short for recipe)
Bacto-Agar (for plates)
Dextrose (Fisher Scientific, catalog number: D16 )
Note: Make 20% stock in double distilled water.
Yeast Nitrogen base w/o amino acids w/ammonium sulfate (BD, catalog number: 291940 )
All amino acids, purines and pyrimidines (any source)
SD-complete + 5’ FOA (ura3∆ yeast selection plates) (see http://cshprotocols.cshlp.org/content/2016/6/pdb.rec086637.short for recipe).
Bacto-Agar
Dextrose (Fisher Scientific, catalog number: D16 )
Note: Make 20% stock in double distilled water.
Uracil
Yeast Nitrogen base w/o amino acids w/ammonium sulfate (BD, catalog number: 291940 )
5-Fluoroorotic acid (5-FOA) (Oakwood Products, catalog number: 003234 )
Equipment
Pipettes (capable of accurately pipetting from 1 µl-10 ml) (any source)
Thermocycler (MJ Research, model: PTC-200 )
Vortex (any source)
Micro centrifuge capable of 14 kG (Eppendorf)
Heating blocks (37 °C, 44 °C)
Incubators (30 °C, 37 °C)
DNA gel electrophoresis apparatus
UV transilluminator (or handheld UV lamp)
Shaker incubator (or rolling drum) (30 °C, 37 °C)
Apparatus for media sterilization (autoclave or pressure cooker)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Dean, N. and Ng, H. (2018). Method for CRISPR/Cas9 Mutagenesis in Candida albicans. Bio-protocol 8(8): e2814. DOI: 10.21769/BioProtoc.2814.
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Category
Microbiology > Microbial genetics > Mutagenesis
Molecular Biology > DNA > Chromosome engineering
Cell Biology > Cell engineering > CRISPR-cas9
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2,815 | https://bio-protocol.org/exchange/protocoldetail?id=2815&type=0 | # Bio-Protocol Content
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Peer-reviewed
3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells
LL Lingli Long
MY Mingzhu Yin
Wang Min
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2815 Views: 10032
Edited by: Jia Li
Reviewed by: Lucíola Silva Barcelos
Original Research Article:
The authors used this protocol in Nov 2016
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Abstract
Ovarian cancer is fairly unique in that ovarian carcinoma cells can detach and spread directly through peritoneal cavity. It has been unclear, however, how detached cancer cells survive in the peritoneum and form spheroid structure. We have recently reported that there is a strong correlation between Tumor-associated macrophages (TAMs)-associated spheroid and clinical pathology of ovarian cancer, and that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in orthotopic mouse models. We have established an in vitro spheroid formation assay using a 3D co-culture system in which mouse GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice were mixed with ID8 cells (TAM:ID8 at a ratio of 1:10) in medium containing 2% Matrigel and seeded onto the 24-well plate precoated with Matrigel. As transcoelomic metastasis is also associated with many other cancers such as pancreatic and colon cancers, TAM-mediated spheroid formation assay would provide a useful approach to define the molecular mechanism and therapeutic targets for ovarian cancer and other transcoelomic metastasis cancers.
Keywords: Ovarian cancer Tumor-associated macrophage 3D co-culture system Spheroid formation Transcoelomic metastasis
Background
Ovarian cancer (OC) is the second most common gynecological cancer and the leading cause of death in the United States (Jemal et al., 2009; Siegel et al., 2012). The major reason for the poor prognosis of OC is intraperitoneal and pelvic extensive implantation metastasis, which is usually unable to be removed completely by surgery. The most widely ascribed explanation for the phenomenon of peritoneal metastasis is that tumor cells become detached from the primary tumor after extension into the peritoneal surface and are transported throughout the peritoneal cavity by peritoneal fluid before seeding intraperitoneally. It has been suggested that the process of transcoelomic metastasis could be divided into several steps: 1) cell detachment, survival and resistance of anoikis; 2) evasion of immunological surveillance; 3) epithelial-mesenchymal transition; 4) spheroid formation; 5) ascites formation; and 6) peritoneal implantation (Tan et al., 2006; Peart et al., 2015; Rafehi et al., 2016). However, it remains unclear how free detached tumor cells survive in transcoelomic environment and form spheroids at initial steps of transcoelomic metastasis. Our recent study reveals that TAMs play an essential role in the survival and proliferation of free cells detached from the primary tumor in transcoelomic environment and spheroid formation at early stages of transcoelomic metastasis (Yin et al., 2016).
One critical method in this study is an in vitro spheroid formation assay using a 3D co-culture system to determine how TAMs facilitate spheroid formation. In this assay, mouse GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice were mixed with ID8 cells (TAM:ID8 at a ratio of 1:10) in medium containing 2% Matrigel and seeded onto the 24-well plate precoated with Matrigel. Similarly, we use human CD14+ TAMs isolated from OC patients and human ovarian cancer SKOV3 cells. In this model, we detect spheroid formation at 48 h of co-culture (Figure 1).
Here, we summarize our detailed protocols for 3D spheroid formation assay.
Figure 1. TAMs and OC cells in vitro 3D co-culture system were showed by Immunofluorescence. A. TAMs and OC cells form spheroids in an in vitro 3D co-culture system. GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice and ID8 cells were co-cultured in the Matrigel-precoated 24-well plate for 48 h. The spheroids were subjected to immunofluorescent staining for E-cadherin for tumor cells. Images for GFP+ TAMs, E-Cadherin+ OC cells and DAPI for all cells in the spheroids are shown. B. Human TAMs were isolated and infected with lentivirus expressing RFP. RFP-expressing TAMs were incubated with SKOV3 human ovarian cancer cells followed by 3D co-culture for 72 h. Spheroids were immunostained with keratin-14. Scale bars = 10 μm.
Materials and Reagents
Pipette tips
15 cm Petri dish
10 ml serological pipette
50 ml sterile conical tube (Corning, Falcon®, catalog number: 352070 )
18-gauge needle
10 ml syringe
FalconTM cell strainers,100 μm (Corning, Falcon®, catalog number: 352360 )
1.5 ml microcentrifuge tubes
Greiner cellstar multiwall culture plates (24 wells, Greiner Bio One International, catalog number: 662102 )
Cell lines: ID8 ovarian cancer cell line (Yin et al., 2016) was a gift from Jack Lawler and Carmelo Nucera at Beth Israel Deaconess Medical Center (Harvard Medical School, Boston, Massachusetts, USA).
Note: ID8 cells are mouse epithelial OC line derived from C57BL/6 background; Passage under 30 and less than 1 week culture before injections.
C57BL/6 mice, female, age: 8 weeks
Tomato reporter transgenic mice: ID8 OC cells were labeled by stably expressing mCherry fluorescence protein while LysMCre mice crossed to the tomato reporter mT/mG
Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 10567014 )
D-glucose
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
0.25% Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
Ketamine
Bovine serum albumin (BSA) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B14 )
Collagenase (Thermo Fisher Scientific, GibcoTM, catalog number: 17104019 )
F4/80 monoclonal antibody, APC conjugate for flow cytometry (Thermo Fisher Scientific, Invitrogen, catalog number: MF48005 )
PE anti-mouse CD206 (MMR) antibody (BioLegend, catalog number: 141705 )
DAPI (Vector Laboratories, catalog number: H-1200 )
Anti-mouse E-Cadherin antibody (BD, PharmingenTM, catalog number: 610404 )
Donkey anti-mouse (Alexa Flour 594) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21203 )
Live cell tracker CMFDA (Thermo Fisher Scientific, InvitrogenTM, catalog number: C2925 )
EGFP (abm, catalog number: LV011-a )
Na2HPO4
NaCl
KH2PO4
KCl
Corning Matrigel (Basement membrane matrix, Corning, catalog number: 356234 )
Tween 20 (Sigma-Aldrich, catalog number: P7949-500ML )
Paraformaldehyde (Sigma-Aldrich, catalog number: 16005 )
PBS (see Recipes)
PBST (see Recipes)
AC buffer (0.5% BSA) (see Recipes)
2% Matrigel (see Recipes)
3.7% paraformaldehyde (see Recipes)
Equipment
Safety cabinet
Pipette-aid
Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
Water bath set at 37 °C
Humidified cell culture incubator set to 37 °C and 5% CO2
Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss, model: Axiovert 200 )
Upright microscope with 10x objective
Cell sorter and the scale (BD, model: FACSAriaTM II ); sorting at a rate of 80,000 cell/h
Software
Quantitation of the average distance between the geometrical center of nuclei of adjacent cells can be measured using Openlab3 software (Improvision, Lexington, MA) or other commercially available image analysis software
SAS software (version 9.1.4, SAS Institute, Cary, NC)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Long, L., Yin, M. and Min, W. (2018). 3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells. Bio-protocol 8(8): e2815. DOI: 10.21769/BioProtoc.2815.
Download Citation in RIS Format
Category
Immunology > Immune cell function > Macrophage
Cancer Biology > Tumor immunology > Cell biology assays
Cell Biology > Cell isolation and culture > 3D cell culture
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2,816 | https://bio-protocol.org/exchange/protocoldetail?id=2816&type=0 | # Bio-Protocol Content
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Peer-reviewed
FACS-based Glucose Uptake Assay of Mouse Embryonic Fibroblasts and Breast Cancer Cells Using 2-NBDG Probe
SD Shengli Dong
Suresh K Alahari
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2816 Views: 10056
Reviewed by: Aswad KhadilkarChristopher J. Poon
Original Research Article:
The authors used this protocol in Oct 2017
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Abstract
This is a flow cytometry-based protocol to measure glucose uptake of mouse embryonic fibroblasts (MEFs) and breast cancer cells in vitro. The method is a slightly modified and updated version as previously described (Dong et al., 2017). Briefly, the target cells are incubated with the fluorescently tagged 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) for 2 h or 30 min, and the efficiency of glucose uptake is examined using a flow cytometer. This method can be adapted to measure a variety of adipocytes, immune cells, MEFs and cancer cells.
Keywords: Mouse embryonic fibroblasts (MEFs) Glucose uptake 2-NBDG Cancer cells Flow cytometry
Background
Glucose is the primary source of energy for cells. A family of glucose transporters (GLUT) is responsible for transporting glucose across cell membranes (Kohn et al., 1996). Changes in glucose uptake can reflect the changes in cellular metabolism. For example, tumor cells generally use glucose for aerobic glycolysis in order to support their rapid proliferation. Normally, tumor cells have increased rates of glucose uptake compared to normal cells (Vander Heiden et al., 2009). The 2-deoxyglucose (2DG) is a glucose analog and it accumulates in the cell as 2-deoxyglucose-6-phosphate (2DG6P). 2DG6P has been a gold standard for measuring glucose uptake for a long time (Yamamoto et al., 2011). Although the measurement of radio-labeled 2DG6P is sensitive, many researchers avoid this method because the handling and disposal of radioactive material require a special procedure.
Another non-metabolizable glucose analog is the fluorescently tagged 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). This molecule accumulates in living cells through a glucose transporter and does not enter the glycolytic pathway. Fluorescence generated by 2-NBDG is proportional to glucose uptake. 2-NBDG fluorescence typically displays excitation/emission maxima of ~465/540 nm. It can be detected using optical filters designed for fluorescein using flow cytometry (O'Neil et al., 2005; Zou et al., 2005; Nitin et al., 2009).
Materials and Reagents
Pipette tips 200 μl tips (USA Scientific, catalog number: 1111-1800 )
10 cm Petri dishes (Corning, catalog number: 430167 )
15 ml polystyrene centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
5 ml round-bottom polystyrene tubes with cell-strainer cap (Corning, Falcon®, catalog number: 352235 ), 0.35 µm nylon mesh
Embryos of C57BL/6 WT mouse (13.5 days)
MCF7 breast cancer cells (ATCC, catalog number: HTB-22 )
Mouse embryonic fibroblasts (MEFs)
0.05% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300062 )
2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) (Thermo Fisher Scientific, InvitrogenTM, catalog number: N13195 )
Dulbecco’s modified Eagle medium (DMEM), high glucose (GE Healthcare, Hyclone, catalog number: SH30243.01 )
Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
Penicillin/streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
2-Mercaptoethanol (2-Me) (Sigma-Aldrich, catalog number: M6250-500ML )
Sodium chloride (NaCl)
Potassium chloride (KCl)
Sodium phosphate dibasic (Na2HPO4)
Potassium phosphate monobasic (KH2PO4)
DMEM culture medium (see Recipes)
Phosphate-buffered saline (PBS, 1x) (see Recipes)
FACS buffer (see Recipes)
Equipment
Pipettes (Gilson, model: P200, catalog number: F123601 )
Refrigerated centrifuge (Eppendorf, model: 5810 R )
Hemocytometer chamber (Hausser Scientific)
Water bath (Fisher Scientific, model: IsotempTM 205 )
Inverted microscope (Nikon Instruments, model: Eclipse Ts2-FL )
Autoclave
Cell culture hood (Thermo Fisher Scientific, Forma class II, A2)
Cell culture incubator (Thermo Fisher Scientific, Forma series II)
FACSCalibur flow cytometer (BD Biosciences)
Software
FlowJo software version 10.0.8 or newer (FlowJo)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cancer Biology > Cellular energetics > Cell biology assays
Biochemistry > Carbohydrate > Glucose
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2,817 | https://bio-protocol.org/exchange/protocoldetail?id=2817&type=0 | # Bio-Protocol Content
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Peer-reviewed
Generation of Luciferase-expressing Tumor Cell Lines
TB Todd V. Brennan
LL Liwen Lin
XH Xiaopei Huang
YY Yiping Yang
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2817 Views: 18118
Edited by: Jia Li
Reviewed by: Shalini Low-NamShweta Garg
Original Research Article:
The authors used this protocol in Jan 2016
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Original research article
The authors used this protocol in:
Jan 2016
Abstract
Murine tumor models have been critical to advances in our knowledge of tumor physiology and for the development of effective tumor therapies. Essential to these studies is the ability to both track tumor development and quantify tumor burden in vivo. For this purpose, the introduction of genes that confer tumors with bioluminescent properties has been a critical advance for oncologic studies in rodents. Methods of introducing bioluminescent genes, such as firefly luciferase, by viral transduction has allowed for the production of tumor cell lines that can be followed in vivo longitudinally over long periods of time. Here we describe methods for the production of stable luciferase expressing tumor cell lines by lentiviral transduction.
Keywords: Lentivirus Tumor Lymphoma Leukemia Luciferase GFP Mouse Methods
Background
Paramount to tracking cells in vivo is the ability to detect them externally by minimally invasive methods. Enzymatic bioluminescence using luciferase derived from the firefly (Photinus pyralis) is a widely used method for image-based cell tracking in vivo. Bioluminescence has been used for a variety of in vivo application including the noninvasive imaging of reporter gene expression (Herschman, 2004), studying circadian rhythms (Southern and Millar, 2005), imaging cerebral strokes (Vandeputte et al., 2014), and for tracking genetically engineered T cells (Costa et al., 2001; Cheadle et al., 2010). Perhaps the field where bioluminescent cell lines have been most applicable is oncology where they have been instrumental for the monitoring tumor growth (Jenkins et al., 2005; Brennan et al., 2016; Byrne et al., 2016) and tumor metastasis (Rosol et al., 2003; Simmons et al., 2015) in mouse models. While some subcutaneously implanted tumors can be detected by palpation and measured with calipers, these methods are not effective for monitoring metastases or tracking tumors that disseminate widely, such as hematological malignancies that commonly grow in the bone marrow, lymph nodes and spleen.
Firefly luciferase oxides luciferin in the presence of molecular oxygen, magnesium and adenosine triphosphate to produce yellow-green light at 560 nm (Wilson and Hastings, 1998; Fraga, 2008). Benefits of luciferase bioluminescence in cell tracking include penetration of tissue for non-invasive monitoring and the re-usability of the enzymatic marker. Another advantage of luciferases is that most cells are not luminescent such that high signal-to-noise ratios can be achieved. A limitation bioluminescence is photon attenuation caused by intervening tissues, such as skin, bone, or hair.
Firefly luciferase is a single polypeptide specified by the luc gene that can be readily cloned into vectors used in gene delivery. Transient expression by plasmid transfection or non-integrating virus transduction limits the time over which cell tracking can be performed. This is especially problematic for oncology studies that may last several months. The ability of retroviruses to integrate into the genome is a key attribute that favors their use in producing stable cell lines. However, some oncoretroviruses, such as the Moloney murine leukemia virus can be limited by transgene silencing over time (Jähner et al., 1982). Further, retroviral vectors require cell division for genomic integration and can be inefficient at transducing highly differentiated cells such as neurons, dendritic cells, or resting lymphocytes.
For the purpose of making luciferase expressing cell lines, lentiviral retroviral vectors derived from human immunodeficiency virus-1 (HIV-1) are highly effective. An advantage of lentiviral vectors over other retroviral vectors, is their ability to integrate into the genome of non-dividing cells. This property makes them suitable gene delivery vehicles for targeting highly differentiated cells, such as neurons, dendritic cells and lymphocytes (Naldini et al., 1996). Lentiviruses also deliver very stable genomic integration and long-term transgene expression, to the extent that they have been used to make transgenic mice following embryo transduction (Lois et al., 2002).
In order to track hematologic tumor cells in an in vivo murine leukemia model, we made the FULGW lentiviral vector that co-expresses firefly luciferase (Luc) and enhanced green fluorescent protein (EGFP) for the purpose of B-cell lymphoma (A20) cell line transduction and stable clone production. The FULGW vector is based on a self-inactivating vector previously described by Miyoshi et al. (1998) that had been engineered to express the GFP reporter gene behind the human ubiquitin-C promoter by Lois et al. (2002), making the FUGW vector. To make FULGW, we replaced the EGFP sequence of FUGW with a Luc-IRES-EGFP sequence from rKat.Luc2.IRES.EGFP, previously developed by Cheadle et al. (2010).
FULGW contains unique elements that enhance gene integration and expression (Figure 1A). It encodes the human immunodeficiency virus-1 (HIV-1) flap element, giving it karyotropic properties that permit efficient genomic integration in non-replicating cells (Zennou et al., 2000). It contains the wood-chuck hepatitis virus posttranscriptional regulatory element (WPRE) that increases gene expression by transcript stabilization (Zufferey et al., 1999). In addition, it includes a 3’ self-inactivating long terminal repeat (3’ si-LTR) that contributes to maintaining it as a replication deficient virus. The 3’ si-LTR was developed by the deletion of a 133 bp region in the U3 region (ΔU3) of the 3’ LTR that renders the 5’ LTR of the integrated provirus transcriptionally inactive (Miyoshi et al., 1998).
Virus production is performed by the co-transfection of HEK-293T cells with the lentiviral plasmid (FULGW) and the two packaging plasmids, pCMV-ΔR8.91 and pCMV-VSVG (Figure 1B). HEK-293T cells are a human embryonic kidney cell line that stably expresses the CMV large T antigen, which greatly increases gene expression by the CMV promoter, generating robust virus production. pCMVΔR8.91 is an HIV-1 Gag and Polymerase (Pol) expression plasmid that was modified from the dR8.9 vector by deletion of four accessory HIV-1 gents, Vif, Vpr, Vpu, and Nef (Zufferey et al., 1997). pCMV-VSVG expresses the pantropic envelop (Env) protein derived from the vesicular stomatitis virus glycoprotein (VSVG) (Stewart et al., 2003). [*Note: Both FULGW and pCMV-ΔR8.91 are large plasmids and best grown in chemically competent recA1-deficient E. coli with high transformation efficiency such as One Shot TOP10 E. coli (Invitrogen) grown at 30 °C for 24-28 h]. Using the FULGW lentiviral vector packaged with these helper plasmids, we have produced multiple types of tumor cell lines on various genetic backgrounds that stably express luciferase and GFP for use in oncologic studies (Table 1).
Figure 1. Production of FULGW lentivirus and transduction of tumor cell lines. A. Diagram of key regions of the FULGW vector including the Luc-IRES-EGFP transgene. Transgene expression is driven by the human ubiquitin-C promoter. CMV (cytomegalovirus promoter), U5 (LTR unique 5’ region), R (LTR repeat region), HIV-1 flap (human immunodeficiency virus-1 flap element), Luc (firefly luciferase), IRES (intra-ribosomal element sequence), EGFP (enhanced green fluorescent protein), WPRE (wood-chuck hepatitis virus posttranscriptional regulatory element), si-LTR (self-inactivating LTR). B. FULGW is packaged and pseudotyped by lipophilic co-transfecting with pCMV-ΔR8.91 and pCMV-VSVG. Virus rich culture supernatant (SN) is collected at 48 and 72 h and virus is concentrated by ultracentrifugation. The concentrated virus is used to transduce tumor cell lines by spin-transduction in the presence of polybrene. Illustrated schematics make use of Motifolio templates (www.motifolio.com/).
Table 1. Luciferase-GFP expressing tumor cell lines produced by FULGW transduction
Materials and Reagents
Pipette tips (USA Scientific, catalog numbers: 1110-3000 , 1110-1000 , 1111-2021 )
T75 flask (Corning, Falcon®, catalog number: 353136 )
100 mm TC-treated Tissue Culture Dish (Corning, Falcon®, catalog number: 353003 )
Sterile syringe 0.45 μm filter (VWR, catalog number: 28145-505 )
Beckman ultra-clear 25 x 89 mm tubes (Beckman Coulter, catalog number: 344060 )
Centricon Plus-70 unit (Merck, catalog number: UFC710008 )
15 ml Falcon tubes (Corning, Falcon®, catalog number: 352099 )
1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5500 )
12-well plates (Corning, catalog number: 3513 )
24-well plates (Corning, Costar®, catalog number: 3526 )
96-well plates (Greiner Bio One International, catalog number: 650185 )
Sterile 500 ml 0.22 μm filter system (Corning, catalog number: 430758 )
29 ga. needles attached to 0.5 ml syringe (Terumo Medical Corporation, Elkton, MD, USA)
BALB/c mice (THE JACKSON LABORATORY, catalog number: 000651 )
HEK 293T (ATCC, catalog number: CRL-3216 )
A20 B-cell lymphoma (ATCC, catalog number: TIB-208 )
pCMV-VSVG plasmid (Addgene, catalog number: 8454 )
pCMVΔR8.91 plasmid (Lifescience Market, catalog number: PVT2323 )
pFULGW (Lentiviral luciferase-IRES-GFP plasmid, Available on request)
Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668027 )
Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11058021 )
Dulbecco’s phosphate buffered saline (DPBS) (Corning, catalog number: 21-031-CM )
Trypsin EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
Polybrene (Sigma-Aldrich, catalog number: TR-1003-G )
Propidium iodide (Sigma-Aldrich, catalog number: P4864-10ML )
Bright-GloTM Luciferase Assay system (Promega, catalog number: E2610 )
D-Luciferin, potassium salt (Gold Bio, catalog number: LUCK-1G )
Isoflurane; Abbott Laboratories (Abbott Park, Illinois, USA)
Fetal bovine serum (Corning, catalog number: 35-010-CV )
DMEM–high glucose (Sigma-Aldrich, catalog number: D6429-500ML )
L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
Gelatin, 2% in H2O, tissue culture grade (Sigma-Aldrich, catalog number: G1393 )
RPMI (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
D10 growth media (see Recipes)
2% gelatin (see Recipes)
D-Luciferin stock solution (see Recipes)
Equipment
Pipettes (Mettler-Toledo, Rainin, catalog numbers: 17008653 , 17008650 , 17008649 ; Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641070N )
Tissue culture hood
Tissue culture incubator (Eppendorf, New Brunswick, model: Galaxy® 170 S )
Fluorescent inverted microscope with GFP filter (Leica Microsystems, model: Leica DM IL LED )
FACS-Canto flow cytometer (BD Biosciences)
Microcentrifuge (Eppendorf, model: 5424 )
Table-top centrifuge (Eppendorf, model: 5810 )
Ultracentrifuge (Beckman Coulter, model: L8-80M ) equipped with an SW-28 rotor (Beckman Coulter, model: SW 28 )
Balance (VWR, catalog number: 10204-990 )
Xenogen IVIS Imaging System (Perkin Elmer, Hopkinton, MA, USA)
Tabletop Laboratory Animal Anesthesia System (VetEquip, catalog number: 901806 )
Autoclave
Software
Acquisition software (CellQuest, BD Biosciences)
Analysis software (FlowJo v9.3, TreeStar)
Living Image Software (Caliper Life Sciences, Hopkinton, MA, USA)
Graphing software (GraphPad Prism v7.0c, La Jolla, CA, USA)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Brennan, T. V., Lin, L., Huang, X. and Yang, Y. (2018). Generation of Luciferase-expressing Tumor Cell Lines. Bio-protocol 8(8): e2817. DOI: 10.21769/BioProtoc.2817.
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Category
Cancer Biology > General technique > Tumor formation
Cell Biology > Cell imaging > Live-cell imaging
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2,818 | https://bio-protocol.org/exchange/protocoldetail?id=2818&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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This is a correction notice. See the corrected protocol.
Peer-reviewed
Correction Notice: Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple
Aparecida L. Silva
KD Keini Dressano
Paulo H. O. Ceciliato
Juan Carlos Guerrero-Abad
Daniel S. Moura
Published: Apr 20, 2018
DOI: 10.21769/BioProtoc.2818 Views: 3410
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In lines 6 and 7, page 4 of our Bio-protocol paper www.bio-protocol.org/e2796, the unit “g” is incorrect, should be “mg”. The same mistake is repeated on page 7, lines 27 and 28. The correct text should be:
Page 4
Procedure
Add 0.006% (w/v) of bromocresol purple free acid reagent grade (60 mg L-1); 1 mM of CaSO4 (172 mg L-1) in sterile distilled water. Adjust pH to 5.7 using KOH or HCl. Pour the solution into an Erlenmeyer.
Page 7
Recipes
Gel containing bromocresol purple (pH 5.7)
0.006% (w/v) bromocresol purple (free acid reagent grade) (60 mg L-1)
1 mM of CaSO4 (172 mg L-1)
Sterile distilled water
Agarose (15 g L-1)
Reference
Silva, A. L., Dressano, K., Ceciliato, P. H., Guerrero-Abad, J. and Moura, D. S. (2018). Evaluation of root pH change through gel containing pH-sensitive indicator bromocresol purple. Bio-protocol 8(7): e2796.
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Silva, A. L., Dressano, K., Ceciliato, P. H. O., Guerrero-Abad, J. C. and Moura, D. S. (2018). Correction Notice: Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple. Bio-protocol 8(8): e2818. DOI: 10.21769/BioProtoc.2818.
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2,819 | https://bio-protocol.org/exchange/protocoldetail?id=2819&type=0 | # Bio-Protocol Content
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Peer-reviewed
Magnetic Resonance Imaging and Histopathological Visualization of Human Dural Lymphatic Vessels
SH Seung-Kwon Ha
GN Govind Nair
MA Martina Absinta
NL Nicholas J. Luciano
DR Daniel S. Reich
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2819 Views: 10592
Edited by: Zinan Zhou
Reviewed by: Marc-Antoine Sani
Original Research Article:
The authors used this protocol in Oct 2017
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Original research article
The authors used this protocol in:
Oct 2017
Abstract
In this protocol, we describe a method to visualize and map dural lymphatic vessels in-vivo using magnetic resonance imaging (MRI) and ex-vivo using histopathological techniques. While MRI protocols for routine imaging of meningeal lymphatics include contrast-enhanced T2-FLAIR and T1-weighted black-blood imaging, a more specific 3D mapping of the lymphatic system can be obtained by administering two distinct gadolinium-based MRI contrast agents on different days (gadofosveset and gadobutrol) and subsequently processing images acquired before and after administration of each type of contrast. In addition, we introduce methods for optimal immunostaining of lymphatic and blood vessel markers in human dura mater ex-vivo.
Keywords: Lymphatic vessels Brain Meninges MRI Histopathology Immunohistochemistry
Background
Among the causes of immune privilege in the brain is the absence of parenchymal lymphatic vessels. However, recent studies have uncovered an extensive lymphatic circulating system in the dura mater of rodents (Aspelund et al., 2015; Louveau et al., 2015), providing possible routes for the elimination of the brain’s waste products and for immune cells to access the deep cervical lymph nodes. In this protocol, we describe a way to: (1) visualize the lymphatic vessels in-vivo in the dura mater using MRI of the head, and (2) assess the local presence of lymphatic vessels using optimized immunostaining methods (Absinta et al., 2017). In-vivo imaging of lymphatics may enable more detailed studies of mechanisms of waste removal and immune function and their potential abnormalities in various diseases and aging.
Materials and Reagents
Superfrost Plus Microslides (Daigger Scientific, catalog number: EF15978Z )
Cover Glasses (Daigger Scientific, catalog number: EF15972L )
Paper towel (KCWW, Kimberly-Clack, catalog number: 05511 )
Polypropylene Coplin jar (IHC World, catalog number: IW-2501 )
Super HT PAP pen (Biotium, catalog number: 22006 )
Gadavist, gadobutrol (0.1 mmol/kg body weight, i.v., Bayer Health Care, NDC 50419-325-12)
Ablavar, gadofosveset (0.03 mmol/kg body weight, i.v., Lantheus Medical Imaging, NDC 11994-012-02)
10% Neutral Buffered Formalin Fixatives, methanol < 2% (Leica Biosystems, catalog number: 3800602 )
Ethanol (Pharmaco-AAPER, catalog number: 111000200 )
Target Retrieval Solution, pH 9 (Agilent Technologies, Dako, catalog number: S2367 )
Target Retrieval Solution (Agilent Technologies, Dako, catalog number: S1699 )
Tris buffered saline 10x, pH 7.4 (KD Medical, catalog number: RGF-3385 )
Hydrogen Peroxide, 30% (Fisher Scientific, catalog number: H325-500 )
Protein Block, Serum-Free (Agilent Technologies, Dako, catalog number: X0909 )
LYVE1 antibody (Abcam, catalog number: ab36993 )
Podoplanin (D2-40) antibody (Bio-Rad Laboratories, catalog number: MCA2543 )
CD31 antibody (Abcam, catalog number: ab28364 )
PROX1 antibody (AngioBio, catalog number: 11-002P )
COUP-TF II antibody (R&D Systems, catalog number: PP-H7147-00 )
CCL21 antibody (Abcam, catalog number: ab9851 )
CD68 (KP-1) antibody (Thermo Fisher Scientific, Invitrogen, catalog number: MA5-13324 )
CD3 antibody (Agilent Technologies, Dako, catalog number: A0452 )
Antibody Diluent (Agilent Technologies, Dako, catalog number: S0809 )
PV Poly-HRP Anti-Mouse IgG (Leica Biosystems, catalog number: PV6114 )
PV Poly-HRP Anti-Rabbit IgG (Leica Biosystems, catalog number: PV6119 )
ImmPRESSTM-AP Anti-Rabbit IgG (Vector Laboratories, catalog number: MP-5401 )
ImmPRESSTM-AP Anti-Mouse IgG (Vector Laboratories, catalog number: MP-5402 )
Goat anti-Mouse IgG, Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-11029 )
Goat anti-Rabbit IgG, Alexa Fluor 594 (Thermo Fisher Scientific, Invitrogen, catalog number: A-11012 )
DAB substrate kit (Abcam, catalog number: ab94665 )
Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories, catalog number: SK-5300 )
Hematoxylin 560 MX (Leica Biosystems, catalog number: 3801575 )
Blue buffer 8 (Leica Biosystems, catalog number: 3802916 )
VectaMount Permanent Mounting Medium (Vector Laboratories, catalog number: H-5000 )
Fluoro-Gel II Mounting Medium (Electron Microscopy Sciences, catalog number: 17985-50 )
Tween 20 (Agilent Technologies, Dako, catalog number: S1966 )
TBS-0.5% Tween 20 (TBST) (see Recipes)
Equipment
18-22 gauge catheter (Smiths Medical)
Pressure infusion tubing (ICU Medical)
Automatic pressure injector (Bayer, model: Medrad® Spectris Solaris® EP MR Injection System )
3-tesla MRI scanner unit (Siemens Skyra, Siemens Healthcare)
32-channel head coil for MRI signal reception (Siemens Skyra, Siemens Healthcare)
Water bath (Leica Biosystem, model: Leica HI1210 )
Humidified chamber (Simport, model: StainTrayTM M920 )
Manual Rotary Microtome (Leica Biosystem, model: Leica RM2235 )
Leica RM CoolClampTM (Leica Biosystem, model: Leica RM CoolClamp )
Steamer (IHC World, model: IHC-TekTM Epitope Retrieval Steamer Set )
Digital rocker (VWR, catalog number: 12620-906 )
Microscope (Carl Zeiss, model: AxioObserver Z.1 )
Microscope camera (Carl Zeiss, model: Axiocam 503 )
Magnetic stirrer
Software
MIPAV software (https://mipav.cit.nih.gov/)
OsiriX software (http://www.osirix-viewer.com/)
Zeiss Zen 2 Blue edition (https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html)
Procedure
Ethical approval
All human research was carried out under an Institutional Review Board approved protocol, after obtaining informed consent. Formalin-fixed human dura was retrieved at autopsy after obtaining appropriate consent.
Human imaging
Place an intravenous line using an 18-22 gauge catheter and pressure infusion tubing linked to an MRI compatible automatic pressure injector. (Figure 1)
Figure 1. MRI preparation. Auto injector setup showing the injector (A), linked to the catheter through extension tubing (B, C), and the setup before the subject is moved into the MRI for scanning (D).
Set up the subject in the MRI scanner with a 32-channel head coil.
Perform cranial MRI as following sequences, a quoted method from Absinta et al. (2017).
Whole-brain T1-Magnetization Prepared Rapid Acquisition of Gradient Echoes (MPRAGE, sagittal 3D turbo-fast low angle shot sequence, acquisition matrix 256 x 256, isotropic resolution 1 mm, 176 slices, repetition time [TR]/echo time [TE]/inversion time [TI] = 3,000/3/900 msec, flip angle 9°, acquisition time 5 min 38 sec).
Limited T2-weighted Fluid Attenuation Inversion Recovery (FLAIR, coronal 2D acquisition over the superior sagittal sinus, field-of-view 256 mm2, 22 slices, reconstructed in-plane resolution 0.25 mm2, 42 contiguous 3 mm slices, TR/TE/TI = 6,500/93/2,100 msec, echo train length 17, bandwidth 80 Hz/pixel, acquisition time 5 min), optimized for detection of gadolinium-based contrast agent in the subarachnoid space.
Black-blood scan (coronal acquisition, Sampling Perfection with Application optimized Contrasts using different flip angle Evolution [SPACE] sequence, field-of-view 174 mm2, matrix 320 x 320, reconstructed in-plane resolution 0.27 mm2, 64 contiguous 0.5 mm sections, TR/TE = 938/22 msec, echo train length 35, bandwidth 434 Hz/pixel, acquisition time 7 min 50 sec). Acquire a series of 2 or three overlapping coronal acquisitions to cover most of the cerebral hemispheres.
Whole-brain T2-FLAIR scan (coronal 3D SPACE sequence, field-of-view 235 mm2, matrix 512 x 512, reconstructed in-plane resolution 0.46 mm2, 176 1 mm sections, TR/TE/TI = 4,800/354/1,800 msec, nonselective inversion pulse, echo-train length 298, bandwidth 780 Hz/pixel, acceleration factor 2, acquisition time 14 min).
Whole-brain T1-SPACE (axial 3D acquisition, acquisition matrix 256 x 256, isotropic resolution 0.9 mm, 112 sections, TR/TE = 600/20 msec, flip angle 120°, echo-train length 28, acquisition time 10 min).
Inject MRI contrast agent, either gadobutrol (0.1 mmol/kg body weight, i.v., Bayer HealthCare) or another standard agent, at a rate of 0.3 ml/min followed by 10 ml of saline flush.
Repeat MRI sequences A3a, A3c, and A3d after completion of the infusion.
Covert scanner-generated DICOM images into NIFTI files for processing using dcm2nii script (nitrc.org, open source).
Co-register pre- and post-contrast images, perform skull-stripping, and subtract pre-contrast images from post-contrast images using standard algorithms implemented in MIPAV software (select Algorithms/Registration/Optimized Automatic Registration and Utilities/Image Calculator/Subtract, respectively).
Import subtraction images into OsiriX software for maximum intensity projection (MIP) 3D rendering (select 2D/3D and then 3D Surface Rendering). (Figure 2)
Figure 2. MRI visualization of dural lymphatic vessels in human. On post-gadobutrol coronal T2-FLAIR, the dura does not enhance, and lymphatic vessels (red arrows), running alongside the venous dural sinuses and within the falx cerebri, can be appreciated. 3D rendering, using OsiriX software, of putative dural lymphatics (black) in a 47-year old woman, derived from whole-brain T1-weighted SPACE MRI. (Modified from Figure 1 and Figure S1 in Absinta et al. [2017]. Creative Commons Attribution License)
For more specific lymphatic imaging, perform Steps B1-B8 using gadofosveset (0.03 mmol/kg body weight, i.v., Lantheus Medical Imaging) rather than gadobutrol. Compare the subtraction images obtained from gadofovest and gadobutrol experiments to identify the lymphatic vessels (Figure 3).
Figure 3. Gadobutrol vs. gadofosveset in MRI visualization of dural lymphatic vessels. Coronal T1-weighted black-blood images were acquired after intravenous injection of two different gadolinium-based contrast agents during two MRI sessions separated by one week. Dural lymphatics (red arrows in magnified view boxes) were better discerned using gadobutrol (standard MRI contrast agent, which readily enters the dura) compared to gadofosveset (serum albumin-binding contrast agent, which remains largely intravascular) and were localized around dural sinuses, middle meningeal artery, and cribriform plate (white arrows). Notably, the choroid plexus (white arrows) enhanced less with gadofosveset than gadobutrol, whereas meningeal and parenchymal blood vessels (both veins and arteries) did not enhance with any contrast agent and appeared black. (Originally published in Absinta et al. (2017). Creative Commons Attribution License)
Immunohistochemistry, single staining
Fix freshly dissected human dura mater with 10% formalin for 24-48 h at room temperature. Commercial 10% neutral buffered formalin (NBF) contains a small percentage of methanol as a stabilizer, which is not a problem for the majority of procedures. Dura should be fixed as soon as possible using gentle agitation (swirling) of the specimen to aid penetration and fixation reaction. Tissue should be fixed for 24-48 h in NBF, and then stored in 1x PBS with a few drops of 10% formalin at room temperature.
Trim the dura into coronal sections and embed the tissue in a paraffin block (see Figure 4). Our recommendation is to focus on the coronal sections near the superior sagittal sinus, which can be easily identified in the dura.
Figure 4. Whole-mount and coronal sections of the human dura mater for histological analysis. A. The red dotted line shows the sampling direction. B, C, and D. Show the coronal view of the dura mater sample before tissue processing.
Using rotary microtome, cut the paraffin-embedded tissue block into sections of 3-8 µm thickness. Float the sections in 20% ethanol at room temperature, then transfer them to a 44 °C water bath. (see Note 1 and Video 1)
Video 1. Demonstration of the sectioning of the human dura mater using a microtome. Before sectioning, place the paraffin tissue block surface on melting ice or cold wet paper towel. After sectioning, place the section in 20% ethanol and then into a warm floating bath.
Transfer the sections onto Superfrost Plus Microslides, as uncoated or uncharged slides may not retain the tissue. Before drying out the slides, remove residual water using a snap of the wrist (imagine wielding a whip), which is important to prevent sections from lifting from slides. Allow the slides to dry vertically overnight, at room temperature, to allow trapped water to escape downward.
Deparaffinize slides using xylene (3 changes of xylene, each 3 min).
Rehydrate slides using 100% alcohol (3 changes, each 3 min), 80% alcohol (3 min) and 50% alcohols (3 min), respectively.
Rinse slides in deionized water for 1 min.
Perform heat-induced antigen retrieval to unmask the antigenic epitope using a steamer. Add tap water to the water base, to the “Max” line, and put the steaming plate onto the water reservoir. Fill a plastic Coplin jar with Target Retrieval Solution or Target Retrieval Solution, at pH 9, and dip deparaffinized/rehydrated slides in the jar. Place the plastic Coplin jar in the steamer and cover it. Turn on the steamer and set the timer for 20 min to incubate it at 95-100 °C. We recommend steamer for heat-induced antigen retrieval instead of microwave or pressure cooker, because it reduces the chance of the section falling off the slide.
Take out the Coplin jar and allow it to cool down for 10 min at room temperature.
Rinse slides gently in Tris-buffed saline (TBS) for 5 min. Use TBS or TBS-0.5% Tween 20 (TBST) during slide washing to prevent sections from falling off.
Immerse sections in 0.3% H2O2 solution in deionized water at room temperature for 10 min to block endogenous peroxidase activity.
Rinse slides gently in TBST for 1 min.
Draw the hydrophobic barrier around the tissues using PAP pen.
Rinse slides gently in TBST 20 for 1 min.
Drop 3-4 of Dako Protein Block on the tissue and incubate at room temperature for 20 min in a humidified chamber.
Gently drop off the excess Dako Protein Block from the slides. Do not rinse the slides in this step.
Apply primary antibody + Dako Antibody Diluent (see Table 1 for antibody dilution factor; 100-200 µl is required to cover the tissue) on the tissues, and incubate at room temperature for 2 h or at 4 °C overnight in a humidified chamber. Make sure that the antibody is spread well on the tissues.
Table 1. Condition of antigen retrieval, antibody dilution and time of incubation
Wash slides in TBST 3 times, 5 min each, using a rocker.
Apply secondary antibody (HRP anti-Mouse IgG or HRP anti-rabbit IgG) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
Wash slides in TBST 3 times, 5 min each, using a rocker.
Drip 3-4 drops of freshly made DAB substrate solution on the slide and check the brown color of antibody signal by microscopy.
If the staining reveals adequate intensity, stop the DAB reaction by dipping slides in deionized water. Over-staining will lead to high background that will obscure the true signals.
Dip slides in Leica Hematoxylin 560 MX for 10 sec, for better morphology and contrast.
Rinse slides in tap water for 5 min.
Immerse slides in bluing solution (Leica Blue buffer or 0.2% ammonia solution or 0.1% lithium carbonate solution).
Dehydrate slides through air dry and coverslip using Permount mounting solution. The mounted slides can be kept at room temperature constantly.
Immunohistochemistry, double staining of D2-40 and CD31 (simultaneous double staining of lymphatic and blood vessels, respectively)
Follow Steps C5-C16 above.
Apply cocktails of primary antibodies + Dako Antibody Diluent on the tissues and incubate at room temperature for 2 h in a humidified chamber.
Wash slides in TBST 3 times, 5 min each, using a rocker.
Apply secondary antibody (HRP anti-Mouse IgG for D2-40 and ImmPRESSTM-AP anti-Rabbit IgG for CD31) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
Wash slides in TBST 3 times, 5 min each, using a rocker.
Drip 3-4 drops of freshly made DAB substrate solution on the slide and check the brown color of D2-40 antibody signal by microscopy.
Wash slides in deionized water to stop the DAB reaction.
Drip 3-4 drops of fresh Vector Blue substrate solution on the same slide and check the blue color of CD-31 antibody signal by microscopy.
If the staining reveals enough intensity, stop the Vector Blue reaction by dipping slides in deionized water.
CAUTION: Do NOT perform hematoxylin counterstaining following use of the Vector Blue chromogen.
Dehydrate slides through air dry and coverslip using Permount mounting solution.
Immunohistochemistry, double staining of PROX1 and CD31 (sequential double staining)
Follow Steps C5-C22 above. Finish PROX1 immunostaining without counterstaining.
Drip 3-4 drops of Dako Protein Block on the tissue and incubate at room temperature for 20 min in a humidified chamber.
Gently drop off the excess Dako Protein Block from the slides. Do not rinse the slides in this step.
Apply CD31 antibodies + Dako Antibody Diluent on the tissues and incubate at room temperature for 2 h in a humidified chamber.
Wash slides in TBST 3 times, 5 min each, using a rocker.
Apply secondary antibody (ImmPRESSTM-AP anti-Rabbit IgG for CD31) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
Wash slides in TBST 3 times, 5 min each, using a rocker.
Drip 3-4 drops of fresh Vector Blue substrate solution on the same slides and check the blue color of CD-31 antibody signal by microscopy.
Stop the Vector Blue reaction by dipping slides in deionized water if the staining reveals enough intensity.
CAUTION: Do NOT perform hematoxylin counterstaining following use of the Vector Blue chromogen.
Dehydrate slides through air drying and coverslip using Permount mounting solution.
Immunofluorescence, double staining of D2-40 + CD31 (simultaneous double staining)
Follow Steps D1-D3 above.
Apply cocktails of secondary antibodies (Goat anti-Mouse IgG Alexa Fluor 488 and Goat anti-rabbit IgG Fluor 594, 1:200 diluted in Dako Antibody Diluent) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
Wash slides in TBST 3 times, 5 min each, using a rocker.
Dehydrate slides through air dry and coverslip using Fluoro-Gel II Mounting Medium.
Observe the localization of D2-40 and CD31 with fluorescence microscopy.
Data analysis
Scan the entire slide and stitch it together by greater than 10x magnification using Zeiss Microscope, camera, and Zeiss Zen Blue software. On slides double-stained for lymphatic and vascular endothelial markers (D2-40/CD31 and PROX1/CD31), identify lymphatic structures and mark them on the screen under the microscope using the following criteria: (a) structures of endothelial cell-lined vessel; (b) vessel with thin endothelial cells, the nuclei of cell bulge into the lumen; (c) semi-collapsed thin vessel wall with poor basal lamina; and (d) no or only a few red blood cells in the lumen of the vessel (Killer et al., 2008). Lymphatic vessels are counted, and their dimensions are measured. If samples vary in disease type or treatment status, simple comparative statistics may be computed on the count and diameter data (Figure 5).
Figure 5. Neuropathology of human dural lymphatic vessels, coronal section. A, B and C. Within the dura mater, lymphatic and blood vessels can be differentiated using double staining for PROX1 (a transcription factor involved in lymphangiogenesis, nuclear staining) and CD31 (a vascular endothelial cell marker). E, F and G. Similarly, lymphatic and blood vessels can be differentiated using double staining for D2-40 (endothelial membrane staining) and CD31. Red blood cells are seen within blood vessels, but not within lymphatic vessels. D and H. Using Zeiss Zen Blue software, lymphatic structures are marked on the digitalized slide. Insets (B, C, F, G) were rotated relative to the original Figures in A and E. Scale bars: 1 mm (A, G), 100 μm (B, C, F, G). Abbreviations: LV–lymphatic vessels; BV–blood vessels. (Modified from Figure 3 in Absinta et al. [2017]. Creative Commons Attribution License)
Notes
Human dura mater is a very tough tissue, and microtome sectioning is difficult. Chilling the paraffin blocks (e.g., Leica RM Cool ClampTM) makes sectioning of dura easier. Also, when tissue is exposed on the surface of a paraffin block by rough trimming, it has the capacity to absorb water, which can penetrate a small distance into the tissue, resulting in softening and swelling it. For the dura mater, this effect may allow a couple of sections to be cut easily. By placing the trimmed block surface on melting ice or in a tray of ice water at 4 °C for 1 min, followed by use of a cold wet paper towel for 30 sec to 1 min, the sectioning becomes easier. Generally, after this procedure, the best quality sections are achieved by cutting very slowly.
Paraffin sections of dura may wrinkle easily, which can generate artifacts and ultimately nonspecific staining. Non-standard flotation techniques may be useful if the sections obtained from a block are highly wrinkled. If sections are initially floated in 20% ethanol then transferred, on a slide, to a hot flotation bath, the wrinkling may be mitigated. 20% ethanol actively removes the wrinkles out because it has lower surface tension than water.
Formalin fixed-paraffin embedded (FFPE) human skin can be used as a positive control for lymphatic vessel marker and assessment. FFPE Hippocampus (CA3) of brain tissue can be used as good positive control for PROX1 staining.
Recipes
TBS-0.5% Tween 20 (TBST)
200 ml 10x TBS
1,800 ml deionized water
Add 1 ml of Tween 20, mixed well using a magnetic stirrer
Acknowledgments
The Intramural Research Program of NINDS supported this study. This protocol was adapted from procedures published in Absinta et al. (2017). Figures 2, 3, and 5 were modified and reproduced with permission from Absinta et al. (2017). The authors declare no conflicts of interest.
References
Absinta, M., Ha, S. K., Nair, G., Sati, P., Luciano, N. J., Palisoc, M., Louveau, A., Zaghloul, K. A., Pittaluga, S., Kipnis, J. and Reich, D. S. (2017). Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. eLife: e29738.
Aspelund, A., Antila, S., Proulx, S. T., Karlsen, T. V., Karaman, S., Detmar, M., Wiig, H. and Alitalo, K. (2015). A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med 212(7): 991-999.
Louveau, A., Smirnov, I., Keyes, T. J., Eccles, J. D., Rouhani, S. J., Peske, J. D., Derecki, N. C., Castle, D., Mandell, J. W., Lee, K. S., Harris, T. H. and Kipnis, J. (2015). Structural and functional features of central nervous system lymphatic vessels. Nature 523(7560): 337-341.
Killer, H. E., Jaggi, G. P., Miller, N. R., Flammer, J. and Meyer, P. (2008). Does immunohistochemistry allow easy detection of lymphatics in the optic nerve sheath? J Histochem Cytochem 56(12): 1087-92.
Copyright: Ha et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Ha, S., Nair, G., Absinta, M., Luciano, N. J. and Reich, D. S. (2018). Magnetic Resonance Imaging and Histopathological Visualization of Human Dural Lymphatic Vessels. Bio-protocol 8(8): e2819. DOI: 10.21769/BioProtoc.2819.
Absinta, M., Ha, S. K., Nair, G., Sati, P., Luciano, N. J., Palisoc, M., Louveau, A., Zaghloul, K. A., Pittaluga, S., Kipnis, J. and Reich, D. S. (2017). Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. eLife: e29738.
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Neuroscience > Cellular mechanisms > Lymphatic vessel
Biophysics > NMR spectroscopy > NMR imgaing
Cell Biology > Tissue analysis > Tissue staining
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282 | https://bio-protocol.org/exchange/protocoldetail?id=282&type=0 | # Bio-Protocol Content
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Peer-reviewed
Protein Translation Study – Label Protein with S35 Methionine in Cells
Salma Hasan
Isabelle Plo
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.282 Views: 27706
Original Research Article:
The authors used this protocol in Mar 2012
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Abstract
To follow protein synthesis, cells should be incubated with radioactive amino acid such as [35S] methionine during mRNA translation. Then, the neosynthetized protein will be identified by an autoradiography after immunoprecipitation with a specific antibody and separation on a polyacrylamide denaturing gel.
Keywords: Protein synthesis Translation S35 methionine
Materials and Reagents
Methionine-free medium DMEM (Sigma-Aldrich, catalog number: D0422 )
Fetal calf serum (Hyclone, catalog number: SV30160.03 )
Fetal bovine serum (FBS)
Penicillin/streptomycin/glutamine
Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 10010-056 )
Protein assay kit (DC Protein Assay Kit I-500) (Bio-Rad, catalog number: 0111EDU )
Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )
EasyTaq -[35S]-Methionine, 5 mCi (185 MBq), stabilized aqueous solution (Perkinelmer, catalog number: NEG709A005MC )
Hybond ECL Nitrocellulose Membrane (Amersham, catalog number: RPN68D )
Kodak Biomax XAR film (Sigma-Aldrich, catalog number: F5763 )
Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, catalog number: WBKLS0500 )
Anti-MDM2 (Santa Cruz)
HEPES
NaCl
Glycerol
Triton X-100
MgCl2
EGTA
Na4P2O7
NaF
Aprotinin
Leupeptin
PMSF
Na3VO4
2-mercaptoethanol
Acrylamide
Bromophenol blue
Ammonium persulfate (APS)
Lysis buffer (see Recipes)
HNTG buffer (see Recipes)
Protein A or G agarose beads (see Recipes)
2x laemmli buffer (see Recipes)
SDS-polyacrylamide gel (see Recipes)
10x electrophoresis buffer (see Recipes)
1x transfer buffer (see Recipes)
Equipment
Centrifuges
Vortexer
Tissue culture hood and incubator
Radioactive material and room
Western-Blot apparatus
Developer
Hamilton syringe
Spectrophotometer
Rocker
T25 flask
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hasan, S. and Plo-Azevedo, I. (2012). Protein Translation Study – Label Protein with S35 Methionine in Cells . Bio-protocol 2(21): e282. DOI: 10.21769/BioProtoc.282.
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Biochemistry > Protein > Labeling
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2,820 | https://bio-protocol.org/exchange/protocoldetail?id=2820&type=0 | # Bio-Protocol Content
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Generation of microRNA Sponge Library
Sebastian Herzog
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2820 Views: 6138
Edited by: Alka Mehra
Reviewed by: Antoine de MorreeSmita Nair
Original Research Article:
The authors used this protocol in Sep 2017
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Abstract
This protocol describes the generation and functional validation of microRNA (miRNA) sponge or decoy constructs. When expressed from a strong promoter, these transcripts can sequester specific miRNA:RISC complexes, thereby resulting in a derepression of endogenous target mRNA. Hence, cells expressing such sponges display a partial or full miRNA loss-of-function phenotype.
Depending on the sponge sequence, the activity of any miRNA of choice can be inhibited by sponge sequestration, but it should be noted that these constructs do not seem to be specific for one particular miRNA. Rather, all miRNAs of the same family as defined by the seed sequence will be affected, albeit to a different degree.
Keywords: microRNA miRNA microRNA decoy microRNA sponge microRNA knockdown microRNA inhibition
Background
microRNAs (miRNAs) are short, non-coding RNAs with a size of about 21-24 nucleotides that post-transcriptionally silence the majority of all protein-coding genes in mammals. Since their discovery, more and more studies have clearly identified this regulatory layer as a crucial element for almost all physiological processes. Not surprising in this context, aberrant expression of miRNAs has also been causally linked to several human malignancies including cancer.
Using classic gain-of-function approaches, early studies have often utilized overexpression to evaluate the function of a particular miRNA. However, this can reach miRNA levels up to 100-fold or higher compared to the physiological context and may generate a phenotype that is not necessarily linked to the miRNA’s normal function.
To avoid this obvious problem with possible overexpression artifacts, in the last years several techniques for miRNA inhibition have been developed both for in vitro and in vivo use, thereby allowing the analysis of a specific miRNA or a group of miRNAs in a loss-of-function approach. These include e.g., the expression of miRNA decoys or sponges, long transcripts that interfere with miRNA function by sequestering miRNA:RISC complexes in a sequence-specific manner (Ebert et al., 2007; Gentner et al., 2009). Another frequently used strategy to interfere with miRNA function are antagomirs, small antisense RNAs complementary to specific miRNAs that can be brought into cells by different means such as by transfection or by viral transduction (Krützfeldt et al., 2005; Scherr et al., 2007). Notably, antagomirs have also been modified in a way that facilitates their cellular uptake, making them an attractive therapeutic tool. More recently, CRISPR/Cas9-mediated genome editing has been shown to be able to abolish miRNA expression on the level of its gene (Chang et al., 2016).
Here I provide a detailed protocol for the generation and functional validation of microRNA sponges as recently described (Lindner et al., 2017). Compared to synthesized small RNAs such as antagomirs, miRNA sponges are cheap and can be stably expressed by retroviral integration. This allows the long-term miRNA knockdown in almost every type of cell and tissue, even in specimen that are difficult to transfect. Moreover, the expression of the sponge RNA can be linked to a fluorescent and/or genetic marker, allowing the easy tracking of sponge-positive cells e.g., by microscopy or flow cytometry. Last, miRNA sponges have been shown to sequester not single miRNAs, but rather all miRNAs of the same family as defined by their seed sequence, i.e., sponges may uncover redundant roles of miRNAs expressed in a particular tissue.
Materials and Reagents
1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
PCR tubes (SARSTEDT, catalog numbers: 72.985.002 and 65.986.002 )
Chemically competent E. coli cells (home-made or New England Biolabs, catalog number: C2987H )
DNA oligonucleotides, reverse phase cartridge or HPLC-purified (Sigma-Aldrich, catalog number: OLIGO )
PHUSION polymerase (New England Biolabs, catalog number: M0530 )
Deoxynucleotide (dNTP) solution mix (Carl Roth, catalog number: K039.1 )
TOPO PCR Cloning Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: 451245 )
Plasmid Mini Kit (Carl Roth, catalog number: HP29.2 )
Restriction enzymes (New England Biolabs)
CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1231 )
Agarose Gel Extraction Kit (Jena Bioscience, catalog number: PP-202L )
DNA ladder (Promega, catalog number: G5711 )
LE Agarose (Biozym Scientific, catalog number: 840004 )
Tris base (Sigma-Aldrich, catalog number: T1503 )
Glacial acetic acid (Sigma-Aldrich, EMD-Millipore, catalog number: 27225-M )
EDTA, disodium salt (Sigma-Aldrich, catalog number: E5134 )
Deionized water
10x TAE for DNA electrophoresis (see Recipes)
Equipment
Pipettes (Eppendorf, model: Research® Plus, catalog numbers: 3123000918 , 3123000020 )
PCR machine TProfessional Basic (Biometra, catalog number: 070-701 )
Electrophoresis equipment (Bio-Rad Laboratories)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Herzog, S. (2018). Generation of microRNA Sponge Library. Bio-protocol 8(8): e2820. DOI: 10.21769/BioProtoc.2820.
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Category
Molecular Biology > RNA > miRNA interference
Molecular Biology > RNA > miRNA-mRNA interaction
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2,821 | https://bio-protocol.org/exchange/protocoldetail?id=2821&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Method for Extracting the Nuclear Scaffold from the Chromatin Network
JC Junjie Chen
BT Boon Heng Dennis Teo
JL Jinhua Lu
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2821 Views: 6664
Edited by: Manjula Mummadisetti
Reviewed by: Pearl CampbellAmey Redkar
Original Research Article:
The authors used this protocol in Feb 2018
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Feb 2018
Abstract
Each cell contains many large DNA polymers packed in a nucleus of approx. 10 μm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary scaffold that support this network has not been achieved but biochemical and proteomic investigations of the scaffold can still provide important insights into this chromatin-organizing network. However, this demands highly inclusive and reproducible extraction of the nuclear scaffold. We have recently developed a simple protocol for releasing the scaffold components from chromatins. The inclusiveness of the extract was testified by the observation that, upon its extraction from the nuclei, the remaining nuclear chromatins were liberated into extended and often parallel chromatin fibers. Basically, this protocol includes the generation of pure nuclei, treatment of the nuclei with Triton X-100 to generate envelope-depleted nuclei (TxN), and extraction of the nuclei at 500 mM NaCl in a sucrose-containing buffer. This combined extract of TxN is known as TxNE.
Keywords: Nuclei Extract Scaffold Nucleophosmin-1 Chromatin
Background
Chromatins are densely and dynamically compacted in the nuclei through a complex scaffold of proteins and ribonucleoproteins. Unlike the cytoskeletal networks (Fischer and Fowler, 2015), microscopic observation of this nuclear scaffold is technically challenging. This may reflect the dominance of chromatins inside each nucleus with which the scaffold mingles and negotiates in the nucleus. The spherical arrangement of the nucleus also contributes to challenges in imaging such scaffold structures. A major element of the nuclear scaffold is the nuclear lamina (NL) (Gruenbaum and Foisner, 2015). NL covers the surface of the entire nuclear chromatin mass on which the nuclear envelop (NE) attaches. The peripheral surface of the nuclear chromatin mass is characterized by dense heterochromatin (Gruenbaum and Foisner, 2015). Inside the nucleus, one or more nucleoli can be found that occupy significant nuclear regions (Jordan and McGovern, 1981; Shaw and Jordan, 1995; Pederson, 2011). The nucleolar surface is also surrounded by a rim of dense chromatins and potentially functions as an important interior scaffold to support the chromatins (Chen et al., 2018). The morphology of the nucleoli changes dynamically depending on the stage of the cell cycle which can significantly affect the organization of nuclear chromatins (Hernandez-Verdun, 2011; Chen et al., 2018).
The presence of such scaffold structures has been extensively investigated in 1980-90s although a direct visualization has not been achieved (Gasser, 2002; Laemmli et al., 1992). However, the composition of the nuclear scaffold and interactions among the scaffold elements could be learned through biochemical, proteomic and imaging studies. To this end, effective and reproducible extraction of the nuclear scaffold from the chromatin network is essential. We have recently developed a simple protocol for the extraction of nuclear scaffold proteins (Chen et al., 2018).
This protocol was initially developed based on a study showing that isolated nucleoli could be solubilized at 400 mM NaCl (Trimbur and Walsh, 1993). We adopted this condition to solubilize isolated nuclei. The inclusiveness of the extract was testified by the liberation of nuclear chromatins as extended parallel chromatin fibers (Chen et al., 2018). TxNE can be used to study many aspects of the nuclear scaffold.
Materials and Reagents
0.5-10 L pipette tips (Corning, Axygen®, catalog number: T-300 )
10-200 ml pipette tips (Greiner Bio One International, catalog number: 739290 )
100-1,000 ml pipette tip (Greiner Bio One International, catalog number: 686290 )
T 175 cell culture flask (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 159910 )
T 75 cell culture flasks (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156499 )
150 mm circular tissue culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 168381 )
50 ml Falcon tubes (Greiner Bio One International, catalog number: 227261 )
5 ml blunt-ended syringes (5 ml/cc, Luer slip, Cellotron, https://dcellotron.en.ec21.com/)
15 ml Falcon tubes (Greiner Bio One International, catalog number: 188271 )
Nalgene Oak Ridge High-Speed Centrifuge Tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3114-0030 )
Regular 1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
Low adhesion microcentrifuge tubes (Eppendorf, catalog number: 022431081 )
12 mm glass coverslips (Electron Microscopy Sciences, catalog number: 72231-10 )
Glass slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6776215 )
50 ml syringes (Terumo, catalog number: SS*50LE )
0.22 μm filters (Merck, catalog number: SLGP033RS )
HeLa S3 human cervical carcinoma cells (ATCC, catalog number: CCL-2 )
Trypsin solution (0.05% w/v, Capricorn scientific, catalog number: TRY-1B10 )
0.5% Trypsin (Trypsin-EDTA, 10x stock, Thermo Fisher Scientific, GibcoTM, catalog number: 15400054 )
Protease inhibitors PMSF (BDH, catalog number: 442172C )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
5 M NaCl
1% (w/v) paraformaldehyde
Mouse anti-nucleophosmin-1 (NPM1) antibody (Sigma-Aldrich, catalog number: B0556 )
Rabbit anti-lamin B1 (LB1) antibody (Abcam, catalog number: ab16048 )
Rabbit anti-histone H1.2 (Abcam, catalog number: ab17677 )
Rabbit anti-lamin A/C (Santa Cruz Biotechnology, catalog number: sc-7292 )
Rabbit anti-H1.x antibody (Abcam, catalog number: ab31972 )
Goat anti-mouse IgG (Cy3-conjugated) (Jackson ImmunoResearch Laboratories, catalog number: 115-165-003 )
Goat anti-rabbit IgG (Alexa Fluor 488-conjugated) (Jackson ImmunoResearch Laboratories, catalog number: 115-545-003 )
Mounting medium (Vector laboratories, catalog number: H-1200 )
Any clear Nail polish (We use Daiso Japan Nail Top Coat Clear, 10 ml bottle, made in Taiwan)
12.5% (w/v) gels
Sodium chloride (NaCl) (Merck)
Potassium chloride GR (KCl)(Merck)
Di-sodium hydrogen phosphate (Merck)
Sucrose (First BASE Laboratories, catalog number: BIO-1090-1kg )
Sucrose (Merck)
Tris (Merck)
Tris base (Avantor Performance Materials, J.T. Baker, catalog number: 4109-02 )
Magnesium chloride hexahydrate (Merck, catalog number: 1058331000 )
Magnesium chloride hexahydrate (MgCl2·H2O)( Merck)
DMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 12430054 )
Fetal bovine serum (FBS, GE Healthcare, HyClone, catalog number: SV30160.03 )
Note: We purchased FBS and generated heat-inactivated FBS by incubation at 56 °C for 30 min.
Penicillin-Streptomycin (100x stock, PAN Biotech, catalog number: P06-07100 )
L-glutamine (200 mM, 100x stock, Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
PBS, pH 7.4 (see Recipes)
0.25 M sucrose buffer, pH 7.4 (see Recipes)
2.2 M sucrose buffer, pH 7.4 (see Recipes)
DMEM culture medium (see Recipes)
Equipment
1,000 L pipette (Eppendorf, model: Research® plus, catalog number: 3120000062 )
200 L pipette (Eppendorf, model: Research® plus, catalog number: 3120000054 )
Humidified cell incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Steri-CycleTM CO2 incubator , catalog number: 371)
Homogenizer (Isobiotec Precision Engineering, Heidelberg, Germany)
Tabletop microcentrifuge (Beckman Coulter, model: Microfuge 22R centrifuge , catalog number: 368831)
Tabletop swing bucket centrifuge (Eppendorf, model: 5810 R , catalog number: 5811000320)
Beckman Coulter centrifuge with a JA-20 rotor (Beckman Coulter, model: Avanti J-25 , catalog number: 363102)
BSL-2 cell culture cabinet (Gelman, model: Bioessential Class II series Type A2 Laminar flow BSC, http://gelmansingapore.com/product/bioessential-class-ii-type-a2-series-laminar-flow-biological-safety-cabinets/)
FluoView FV1000 confocal microscope
Cool/SNAP HQ2 image acquisition camera (Olympus)
Software
FV-ASW 1.6b software
Imaris software (Bitplane AG)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Chen, J., Teo, B. H. D. and Lu, J. (2018). A Method for Extracting the Nuclear Scaffold from the Chromatin Network. Bio-protocol 8(8): e2821. DOI: 10.21769/BioProtoc.2821.
Chen, J., Teo, B. H. D., Cai, Y., Wee, S. Y. K. and Lu, J. (2018). The linker histone H1.2 is a novel component of the nucleolar organizer regions. J Biol Chem 293(7): 2358-2369.
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Category
Cell Biology > Cell structure > Nucleus
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2,822 | https://bio-protocol.org/exchange/protocoldetail?id=2822&type=0 | # Bio-Protocol Content
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Peer-reviewed
Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast
Isabel Fernández de Castro
Cristina Risco
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2822 Views: 6009
Edited by: David Paul
Reviewed by: Mirko Cortese
Original Research Article:
The authors used this protocol in Jan 2017
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Jan 2017
Abstract
Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication organelles. Therefore, efficient virus replication depends on the assembly of specialized membranes where viral macromolecular complexes are turned on and hold a variety of functions. Detailed characterization of viral replication platforms in cells requires sophisticated imaging approaches. Here we present a protocol to visualize the three-dimensional organization of the tombusvirus replicase complex in yeast with MEtal-Tagging Transmission Electron Microscopy (METTEM). This protocol allowed us to image the intracellular distribution of the viral replicase molecules in three-dimensions with METTEM and electron tomography. Our study showed how viral replicase molecules build replication complexes within specialized cell membranes.
Keywords: Metal-tagging transmission electron microscopy METTEM Clonable tag Viral replication complex Electron tomography 3D electron microscopy
Background
Replication of positive-stranded RNA viruses depends on the remodeling of cellular membranes. Intracellular membranes serve as a structural scaffold for VRC assembly, provide essential lipids and co-factors that modulate the activity of the viral replicase and protect the viral RNA from the antiviral defenses of the host (Miller and Krijnse-Locker, 2008; den Boon et al., 2010; Nagy and Pogany, 2011; Nagy, 2016). The architecture of replication organelles with active VRCs has been observed by electron microscopy. VRCs assemble in single membrane vesicles or ‘spherules’, tubulovesicular cubic membranes, double membrane vesicles (DMV) or planar oligomeric arrays (de Castro et al., 2013). Spherules are often observed in cells infected by RNA viruses. They form by invagination in a variety of organelles and have a narrow opening to the cytosol (den Boon et al., 2010).
Tomato bushy stunt virus (TBSV) is a small (+) RNA virus that belongs to the Tombusviridae, a family of viruses that infect plants. TBSV has recently emerged as a model virus to study viral replication and virus-host interactions using the yeast Saccharomyces cerevisiae as a model host (Nagy and Pogany, 2011). Studies of S. cerevisiae infected with plant viruses have facilitated the identification of numerous factors needed for viral replication (Nagy, 2008). Tombusviruses encode five proteins including two replication proteins, p92pol and p33. p92pol is the RNA-dependent RNA polymerase. The auxiliary protein p33 is an RNA chaperone that facilitates the recruitment of the viral RNA to the site of replication, in the cytosolic face of peroxisome membranes (McCartney et al., 2005; Jonczyk et al., 2007).
Understanding the biogenesis and functional architecture of VRCs in cell membranes is challenging and it requires sophisticated imaging techniques. Transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of macromolecular assemblies in cells. However, methods to unambiguously identify proteins within the environment of the cell are lagging behind. In our lab, we have developed a new labeling method named METTEM from MEtal-Tagging Transmission Electron Microscopy. This method uses the metal-binding protein metallothionein (MT) as a genetically clonable tag for electron microscopy (Diestra et al., 2009; Risco et al., 2012). Mouse MT 1 is a small, 61-amino acid protein with 20 cysteine residues that bind gold atoms very efficiently. MT fused to a protein of interest and treated with gold salts, builds an electron-dense gold nanocluster of around 1 nm diameter, easily visualized by electron microscopy (Mercogliano and DeRosier, 2006 and 2007) (Figure 1). METTEM allows identification and localization of intracellular proteins with high specificity and exceptional sensitivity at molecular-scale resolution ( Diestra et al., 2009; Delebecque et al., 2011; Bouchet-Marquis et al., 2012; Risco et al., 2012; Barajas et al., 2014a; de Castro Martin et al., 2017; Fernandez de Castro et al., 2017).
Figure 1. Imaging viral replicase complexes with METTEM. Viral replicase protein is fused with metallothionein (MT) and expressed in yeast cells (Fernandez de Castro et al., 2017). MT-tagged viral replicase molecules assemble VRCs in peroxisome membranes. Cells are incubated with gold salts in vivo and MT-gold-replicase molecules are visualized by electron microscopy.
Here we described a protocol to visualize TBSV replicase molecules in VRCs using METTEM (Figure 2). The combination of this technology with electron tomography allowed us to study the distribution of replicase molecules in the viral replication compartment in three-dimensions (3D). Due to the high sensitivity of the method we could distinguish different states of aggregation of the viral replicase molecules in situ. This methodology can be used to detect any protein of interest in different subcellular locations of bacteria, yeast and mammalian cells. Furthermore, one advantage of this electron microscopy approach is that it can be used to study many different viruses in a variety of cell types by visualizing the MT tag incorporated in either complete viral particles or their proteins. This method has revealed virus-induced structures not seen before, as reported for Rubella virus, Tombusvirus and influenza virus (Risco et al., 2012; de Castro Martin et al., 2017; Fernandez de Castro et al., 2014 and 2017).
Figure 2. Schematic workflow of the protocol. Pre-grown transformed yeast cells are incubated overnight in YPG. Next day viral replication is induced during 24 h at 23 °C. Cells are treated with zymolyase to obtain spheroplasts. Spheroplasts are incubated with gold salts to build nanoclusters in MT tags. Cell pellets are dehydrated and embedded in resin. Serial sections are transferred to EM grids and imaged by TEM.
Materials and Reagents
Pipette tips
Perfect loop (Electron Microscopy Sciences, catalog number: 70945 )
50 ml disposable centrifuge tubes
Eppendorf tubes
Sterile transfer pipettes
Serum Acrodisc® 37 mm syringe filter (Pall, catalog number: 4525 )
Gelatin capsule size 1 6.5 mm diameter-0.50 ml (TAAB, catalog number: C089/1 )
GEM® Single Edge Blades 3-Facet 0.009"/0.23 mm (AccuTec Blades, catalog number: 62-0179-0000 )
Saccharomyces cerevisiae yeast strains RS453 (MATa ade2-1 his3, 15 leu2-3, 112 trp1-1 ura3 52) and pah1Δnem1Δ (SwissProt ID for Nem1 is P38757) (pah1Δ::TRP1nem1Δ::HIS3 derivative of RS453) (Choi et al., 2011; Barajas et al., 2014b)
Zymolyase® 20T (Arthrobacter luteus) (Amsbio, catalog number: 120491-1 )
Gold(III) chloride ≥ 99.99% (Sigma-Aldrich, catalog number: 379948 )
Glutaraldehyde 50% (TAAB, catalog number: G015 )
Ethanol Dry (Merck, catalog number: 1009901001 )
LR-White Resin Medium Grade Acrylic resin (TAAB, catalog number: L012 )
BactoTM yeast extract (BD, BactoTM, catalog number: 212750 )
BactoTM peptone (BD, BactoTM, catalog number: 211677 )
D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 )
Agar (Sigma-Aldrich, catalog number: 05038 )
D-(+)-Galactose (Sigma-Aldrich, catalog number: G0750 )
Lithium acetate 99.95% (Sigma-Aldrich, catalog number: 517992 )
ssDNA (Deoxyribonucleic acid sodium salt from salmon testes) (Sigma-Aldrich, catalog number: D1626 )
PEG MW3350 (Polyethylene glycol) (Sigma-Aldrich, catalog number: P4338 )
Trizma® hemisulfate (Sigma-Aldrich, catalog number: T8379 )
1,4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: DTT-RO)
Manufacturer: Roche Diagnostics, catalog number: 10197777001 .
Yeast nitrogen base without amino acids (Sigma-Aldrich, catalog number: Y0626 )
Yeast Synthetic Drop-out Medium Supplements without tryptophan (Sigma-Aldrich, catalog number: Y1876 )
D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
Tris (Base) (Norgen Biotek, catalog number: 28029 )
Paraformaldehyde EM (TAAB, catalog number: P026 )
NaOH
10x PBS
1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N’-bis(2-ethanesulfonic acid) PIPES (Sigma-Aldrich, catalog number: P6757 )
4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
YPD (see Recipes)
YPG (see Recipes)
Transformation mix (see Recipes)
TSD reduction buffer (see Recipes)
Spheroplast medium A (see Recipes)
4% paraformaldehyde (PFA) solution (see Recipes)
PHEM solution, pH 6.9 (see Recipes)
Equipment
500 ml flask
Pipettes
Diamond Knife 45° DIATOME (Fedelco)
QUANTIFOIL® R 3.5/1 100 Holey Carbon Films Grids Au 300 mesh (Quantifoil)
Oven Memmert UN 55 (Memmert, model: UN55 ) (Genesys) equipped with a shaker
Spectrophotometer 722N (Terra Universal, Laboratory Equipment, model: 722N )
Centrifuge 5810 R (Eppendorf, model: 5810 R )
Centrifuge miniSpin plus (Eppendorf, model: MiniSpin® plus )
Ultrasonic Cleaner 1510 (Branson, model: 1510 )
pH meter Basic 20 (HACH LANGE SPAIN, Crison, model: Basic 20 )
Fume Hood (Flow-Tronic)
Ultramicrotome (Leica Microsystems, model: Leica EM UC6 )
Jeol JEM 1011 electron microscope operating at 100 kV (JEOL, model: JEM-1011 )
FEI Tecnai G2 F20 (200 kV) electron microscope (FEI)
Tecnai Spirit Twin (120 kV) electron microscope (FEI)
Software
IMOD software
Amira software
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Fernandez de Castro, I. and Risco, C. (2018). Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast. Bio-protocol 8(8): e2822. DOI: 10.21769/BioProtoc.2822.
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Category
Microbiology > Microbe-host interactions > Virus
Cell Biology > Cell imaging > Electron microscopy
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2,823 | https://bio-protocol.org/exchange/protocoldetail?id=2823&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation and Maintenance of Murine Embryonic Striatal Neurons
LN Luana Naia
AR A. Cristina Rego
Published: Vol 8, Iss 8, Apr 20, 2018
DOI: 10.21769/BioProtoc.2823 Views: 9392
Edited by: Oneil G. Bhalala
Reviewed by: Tatiana Rosado RosenstockRyohei Iwata
Original Research Article:
The authors used this protocol in Sep 2017
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Sep 2017
Abstract
Primary cultures of murine striatal neurons are widely used to explore cellular mechanisms in neurobiology, including brain diseases. Here we describe a detailed and standardized protocol to dissect and culture embryonic murine striatal neurons GABA-positive/DARPP-32-positive for 12 days in vitro, when they show good neuronal cell connectivity and the presence of dendritic spines, which reflects the maturation of the network.
Keywords: Primary cultures Striatum dissection Striatal neurons Medium spiny neurons
Background
Striatum is a critical component of the motor and reward systems and dysfunction of striatal neurons can lead to a variety of neuronal disorders, from obsessive-compulsive-like behaviors (Welch et al., 2007) to neurodegeneration, as observed in Huntington’s disease (Reiner et al., 1988). Therefore, a well-stablished striatal culture may be a great value as a model for studying these and other conditions. The major neuronal subtype in adult striatum is medium spiny projection neurons (MSNs), which constitutes approximately 95% of all striatal neurons and use the inhibitory transmitter gamma-aminobutyric acid (GABA). They are characterized by medium-sized cell bodies, complex dendritic arbors and a high density of dendritic spines that receive both glutamatergic and dopaminergic inputs. The remaining 5% of neurons are composed by GABAergic aspiny interneurons (Graveland and DiFiglia, 1985; Matamales et al., 2009). The following protocol describes the preparation of primary cultures of mouse embryonic striatal neurons with a low percentage of astrocytes, which proliferation is prevented by the mitosis inhibitor 5-fluoro-2’-deoxyuridine (5-FdU). After 12 days in vitro (DIV) the striatal neurons obtained by the described protocol are expected to be immunolabeled for microtubule-associated protein 2 (MAP2) or NeuN, GABA and a large percentage for dopamine- and cAMP-regulated phosphoprotein (DARPP-32).
Materials and Reagents
Coverslips 18 mm diameter round, #1.5 (0.17 mm) thickness (Thermo Fisher Scientific, Menzel, catalog number: 11817742 )
Sterile non-tissue culture treated Petri dishes of 35, 60 and 100 mm (VWR, catalog numbers: 734-1707 , 734-1708 , 734-1709 )
5, 10 ml serological sterile pipettes (Labbox, catalog numbers: MPIP-U05-200 , MPIP-U10-200 )
Sterile 0.2-10 μl, 10-200 μl and 100-1,000 μl micropipette tips (Frilabo, catalog numbers: 170558 , 171017 , 171024 )
15 and 50 ml sterile conical centrifuge tubes (Corning, catalog numbers: 430791 , 430829 )
Sterile 1.5 ml microcentrifuge tubes (BIOplastics, catalog number: B74085 )
Sterile syringes, 20 ml (Terumo, catalog number: SS+20L1 )
Sterile 230 mm glass Pasteur pipettes (Labbox, catalog number: PIPN-230-250 )
Sterile 0.2 μm acetate cellulose filters (GE Healthcare, Whatman, catalog number: 28415732 )
Vacuum 0.2 μm filter bottle system, 500 ml (Corning, catalog number: 430769 )
Cell strainers, 40 μm (Corning, Falcon®, catalog number: 352340 )
Multi-wells tissue culture treated, flat bottom, polystyrene (Corning, catalog numbers: 3596 , 3548 , 3524 , 3512 , 3506 )
Pregnant female mice (Mus musculus) with 16 days of gestation
Note: In this protocol, mice from FVB/NJ inbred strain (THE JACKSON LABORATORY) were used.
Hydrochloric acid (HCl), 37% (Sigma-Aldrich, catalog number: 435570 )
Ethanol absolute 99.8% (Fisher Scientific, catalog number: 10342652 )
Isoflurane Iso-Vet 1,000 mg/g Inhalation Vapour (Chanelle UK)
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
5-FdU (Sigma-Aldrich, catalog number: F0503 )
Anti-MAP2 (1:500; Santa Cruz Biotechnology, catalog number: sc-32791 )
Anti-GABA (1:500; Sigma-Aldrich, catalog number: A2052 )
Anti-DARPP32 (1:100; Abcam, catalog number: ab40801 )
Goat anti-rabbit IgG secondary antibody, Alexa Fluor 594 (1:200; Thermo Fisher Scientific, Invitrogen, catalog number: R37117 )
Donkey anti-mouse IgG secondary antibody, Alexa Fluor 488 (1:200; Thermo Fisher Scientific, Invitrogen, catalog number: R37114 )
Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P1149 )
Potassium chloride (KCl) (Labbox, catalog number: POCL-00P-1K0 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
Sodium chloride (NaCl) (Merck, catalog number: 106404 )
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139 )
D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 )
Sodium pyruvate (100 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Phenol red (Sigma-Aldrich, catalog number: P3532 )
Deoxyribonuclease I (DNase I) (Sigma-Aldrich, catalog number: D5025 )
Bovine serum albumin (BSA) fatty acid free (Sigma-Aldrich, catalog number: A6003 )
Trypsin, Type IV-S, from porcine pancreas (Sigma-Aldrich, catalog number: T0303 )
Trypsin inhibitor, type II-S: Soybean (Sigma-Aldrich, catalog number: T9128 )
L-Glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
Gentamicin (50 mg/ml) (Thermo Fisher Scientific, catalog number: 15750060 )
Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
B-27 supplement (50x), serum free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
Poly-D-lysine for coating (0.1 mg/ml) (see Recipes)
Hanks’ balanced salt solution (HBSS) (see Recipes)
DNase I solution (5 mg/ml) (see Recipes)
BSA solution (3 mg/ml) (see Recipes)
Trypsin solution (0.5 mg/ml) (see Recipes)
Trypsin inhibitor solution (1 mg/ml) (see Recipes)
Washing solution (see Recipes)
Inactivated FBS (see Recipes)
Supplemented Neurobasal medium (see Recipes)
Equipment
11.5 cm fine straight scissor (Fine Science Tools, Dumont, catalog number: 14060-11 )
8.5 cm mini-dissecting scissor, straight with sharp tip (World Precision Instruments, catalog number: 503669 )
Medium forceps (Fine Science Tools, model: Dumont #7, catalog number: 11274-20 )
Forceps with straight and angled fine tip (Fine Science Tools, models: Dumont #5 and Dumont #5/45 )
Automatic pipettor and micropipettes
Glass Schott laboratory bottle with cap, 500 ml (DWK Life Sciences, Duran, catalog number: 21 801 44 5 )
Water bath with electronic temperature regulation
Ultrasonic cleaning bath (just for coverslips washing)
Zeiss Stemi magnification glass (or equivalent)
Phase contrast inverted microscope equipped with 10x and 20x objectives
Zeiss LSM 710 point-scanning confocal microscope (ZEISS, model: LSM 710 or equivalent)
Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
Water jacketed CO2 incubator
Vertical laminar flow chamber
Isoflurane vaporizer apparatus (E-Z Anesthesia, model: EZ-SA800 , or equivalent)
Software
Fiji software (ImageJ, National Institute of Health, USA)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Naia, L. and Rego, A. C. (2018). Isolation and Maintenance of Murine Embryonic Striatal Neurons. Bio-protocol 8(8): e2823. DOI: 10.21769/BioProtoc.2823.
Download Citation in RIS Format
Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Cell isolation and culture
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2,824 | https://bio-protocol.org/exchange/protocoldetail?id=2824&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells
SA Surya Amarachintha
QP Qishen Pang
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2824 Views: 9335
Edited by: Giusy Tornillo
Reviewed by: Nandini Mondal
Original Research Article:
The authors used this protocol in Nov 2015
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Original research article
The authors used this protocol in:
Nov 2015
Abstract
Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.
Keywords: Mesenchymal Stromal Cells Hematopoietic Stem Cells Co-culture assay Cobblestone area-forming cell assay
Background
The proliferative, survival and differentiation potential of HSCs is very much dependent on its microenvironment also known as niche. The bone marrow MSCs support the HSCs to keep them in a quiescent state in the bone marrow niche. The intrinsic and extrinsic signals received by the niche contribute to the differentiation of HSCs into mature blood-cell lineages also known as hematopoiesis, without inducing aberrant expansion (Yoshihara et al., 2007; Spindler et al., 2014; Hu et al., 2016). The Cobblestone-Area-Forming Cell Assay (CAFC Assay) is an in vitro co-culture assay of long-term bone marrow HSCs and MSCs. While MSCs are cultured to complete confluence in a tissue culture dish, HSCs are plated over MSCs (de Haan and Ploemacher, 2002). CAFC assays are comparable to in vivo studies of bone marrow and can be used as a rapid screening assay to test the stem cell activity of HSCs and supportive activity of MSCs (Ploemacher et al., 1989). There is a high demand for high throughput screening models that reflect the complex physiology or pathology of the bone marrow microenvironment both in native state or disease models respectively. In this regard, large scale screening was made possible using the HSCs-stroma co-culture system to identify small-molecule inhibitors to develop an effective therapy for acute leukemia (Hartwell et al., 2013). Further, a co-culture system was applied as a model to study several diseases like Fanconi anemia (FA), where it was identified that FA MSCs produce elevated levels of metabolites like glycerophospholipids, which can skew the normal HSCs function (Amarachintha et al., 2015). Further, MSCs provided an impaired environment for HSCs proliferation in patients with aplastic anemia suffering with pancytopenia and in the patients who are in remission after immunosuppression (Schrezenmeier et al., 1996). Further, MSCs from T-cell lymphocytic leukemia mouse model showed adverse proliferation and differentiation capacity of HSCs (Lim et al., 2016). However, in Cord Blood transplants, Cord Blood-MSC co-culture holds promise for successful expansion of cord blood and surge the engraftment in recipients (Denning-Kendall et al., 2003; Robinson et al., 2006). Although several theories were proposed to characterize the isolation and culture of MSCs, ‘The International Society for Cellular Therapy’ has set the minimal criteria for defining ‘multipotent mesenchymal stromal cells’. MSCs derived from bone marrow must be plastic-adherent in standard culture conditions, express cell surface markers, and must differentiate to osteoblasts, adipocytes, and chondroblasts in vitro (Dominici et al., 2006; Keating, 2012). Adhering to these principles, we identified a simplistic approach to culture MSCs from mouse bone marrow and performed limited dilution CAFC assay to enable rapid screenings.
Materials and Reagents
Pipette tips (USA Scientific, catalog number: 1126-7810 )
1,000 µl large orifice pipette tip (USA Scientific, catalog number: 1011-9000 )
BD Precision glide needles (BD, catalog number: 305155 )
BD Slip Tip Sterile Syringe 1 ml (BD, catalog number: 309659 )
BD Single-use Needles 22 G (BD, catalog number: 305159 )
Falcon 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
Falcon standard tissue culture dishes (100 mm culture dish) (Corning, Falcon®, catalog number: 353003 )
Falcon 35 mm TC-treated Easy-Grip style cell culture dish (Corning, Falcon®, catalog number: 353001 )
Nunc Edge 2.0 96-well cell culture plates (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: 167425 )
3-well chamber slide (IBIDI, catalog number: 80381 )
Falcon Polystyrene Microplates 6-well plate TC-treated (Corning, Falcon®, catalog number: 353934 )
C57BL/6J mice of age 4 to 8 weeks old (THE JACKSON LABORATORY, catalog number: 000664 )
70% ethanol
Red blood cell lysis buffer (Sigma-Aldrich, Roche Diagnostics, catalog number: 11814389001 )
Trypsin-EDTA (0.25%), phenol red (Thermo Fischer Scientific, GibcoTM, catalog number: 25200056 )
Mouse MSC Functional Identification Kit containing antibodies Osteopontin, Fabp4, and Collagen II (R&D Systems, catalog number: SC010 )
DPBS (10x), no calcium, no magnesium (Thermo Fischer Scientific, GibcoTM, catalog number: 14200075 )
16% formaldehyde (w/v), methanol-free (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: 28906 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
4’,6-Diamidino-2-phenylindole (DAPI) (Thermo Fischer Scientific, InvitrogenTM, catalog number: D1306 )
VECTASHIELD Antifade mounting medium (Vector Laboratories, catalog number: H-1000 )
Ficoll-Paque PLUS (GE Healthcare, catalog number: 17144002 )
Streptavidin APC-Cy-7 (BD, BD Biosciences, catalog number: 554063 )
PE rat anti-mouse Ly-6A/E (BD, BD Biosciences, catalog number: 561076 )
Rat anti-mouse CD117 (BD, BD Biosciences, catalog number: 561074 )
V450 mouse lineage antibody cocktail (BD, BD Biosciences, catalog number: 561301 )
Fetal bovine serum, qualified, heat inactivated, USDA-approved (FBS) (Thermo Fischer Scientific, GibcoTM, catalog number: 10438034 )
Iscove’s modified Dulbecco’s medium (Thermo Fischer Scientific, InvitrogenTM, catalog number: 12440053 )
Bovine calf serum (GE Healthcare, Hyclone, catalog number: SH30072.03 )
Epidermal growth factor (R&D Systems, catalog number: 2028-EG-200 )
Platelet-Derived growth factor (R&D Systems, catalog number: 220-BB-010 )
Penicillin-streptomycin (Thermo Fischer Scientific, GibcoTM, catalog number: 15140122 )
2-Mercaptoethanol (Thermo Fischer Scientific, GibcoTM, catalog number: 21985023 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
Donkey serum (Sigma-Aldrich, catalog number: D9663 )
Bone marrow wash buffer (see Recipes)
MSCs media (see Recipes)
DPBS blocking solution (see Recipes)
Equipment
ErgoOne 100-1,000 µl Single Channel Pipettes (USA Scientific, model: 7110-1000 )
Eppendorf 5804 Benchtop Centrifuge (Eppendorf, model: 5804 )
BD FACSCanto II to analyze the hematopoietic stem cells (BD, model: BD FACSCanto II )
BD FACSAria II to sort the hematopoietic stem cells (BD, model: BD FACSAria II )
NIKON Eclipse 90i microscope to capture immunofluorescence images (Nikon Instruments, model: Eclipse 90i )
OLYMPUS IX53 Inverted Microscope to capture phase contrast images of cobblestone area (Olympus, model: IX53 )
Software
BD FACSDiva v8.0.1 Software (BD Biosciences)
FlowJo software (FLOW JO)
CellSens software (Olympus)
GraphPad Prism software
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Amarachintha, S. and Pang, Q. (2018). Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells. Bio-protocol 8(9): e2824. DOI: 10.21769/BioProtoc.2824.
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Category
Stem Cell > Adult stem cell > Hematopoietic stem cell
Cell Biology > Cell isolation and culture > Co-culture
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2,825 | https://bio-protocol.org/exchange/protocoldetail?id=2825&type=0 | # Bio-Protocol Content
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Peer-reviewed
Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies
FM Faruk H. Moonschi
AF Ashley M. Fox-Loe
XF Xu Fu
CR Chris I. Richards
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2825 Views: 6181
Reviewed by: Rahul SrinivasanTalita Diniz Melo Hanchuk
Original Research Article:
The authors used this protocol in Dec 2017
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Abstract
Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.
Keywords: Single molecule Vesicles Stoichiometry ER and plasma membrane protein separation Nicotinic receptor OptiPrep Photobleaching Protein trafficking
Background
A large number of transmembrane proteins are formed through the assembly of multiple subunits leading to complicated oligomeric structures that can often exist in multiple stoichiometries. Understanding how changes in assembly alter trafficking and localization within different organelles is essential to determining a protein’s physiological role and the connection to diseases associated with maturation and transport. Single molecule approaches can provide a better understanding of the assembly of oligomeric proteins by directly measuring their stoichiometry (Ulbrich and Isacoff, 2007; Richards et al., 2012). This approach avoids ensemble averaging which provides the average state of all the stoichiometries (Walter and Bustamante, 2014). Single molecule studies have recently been employed to understand the structural and functional properties of macromolecules including conformational dynamics (Tan et al., 2014), ion channel gating (Wang et al., 2016), ligand-receptor interaction (Moonschi et al., 2015), and stoichiometric assembly (Ulbrich and Isacoff, 2007; Moonschi et al., 2015). To conduct single molecule studies, it is imperative to isolate single receptors in a supporting bilayer. Separation from the cellular environment is often necessary because the endogenous concentration of membrane proteins in mammalian cells is typically much higher than conditions compatible with single molecule studies (Richards et al., 2012). Additionally, live cell single molecule studies suffer from a number of disadvantages including high levels of auto-fluorescence, limited fluorophore brightness and the mobility of membrane proteins on the cell surface (Andersson et al., 1998; Lippincott-Schwartz et al., 1999). Isolation strategies using artificial bilayers such as liposomes are also problematic as they include an intermediate step where the protein is stabilized into a detergent solution which poses a threat to the structural integrity of the transmembrane protein. Membrane-derived vesicles enable the protein to remain embedded in its physiological membrane maintaining its structural and functional integrity. These nanoscale vesicles have been employed to study stoichiometric assembly of multimeric proteins, to probe ligand receptor interactions, and to understand the effect of ligands on the assembly of nicotinic receptors (Fox et al., 2015; Moonschi et al., 2015; Fox-Loe et al., 2017).
Transmembrane proteins are synthesized and assembled in the endoplasmic reticulum and then trafficked to the plasma membrane. Oligomeric proteins with multiple non-identical subunits can often be assembled with different subunit stoichiometries potentially leading to different trafficking and functional properties (Grady et al., 2010). For instance, α4β2 nicotinic receptors are pentameric receptor with two possible stoichiometric assemblies: (α4)2(β2)3 and (α4)3(β2)2. It has been hypothesized that cellular machinery preferentially traffics the high sensitivity isoform (α4)2(β2)3 over the other isoform from the ER to the plasma membrane. It has also been hypothesized that nicotine alters the assembly of this receptor from the low sensitivity to high sensitivity isoform in the ER leading to higher levels of the preferentially trafficked assembly in both the ER and plasma membrane (Lester et al., 2009; Henderson and Lester, 2015). Single molecule studies of proteins from specific organelles enable the correlation of changes in structural assembly of the nicotinic receptors to changes in trafficking and assembly.
Here we present a method which can isolate single transmembrane proteins into the ER and plasma membrane derived vesicles using nitrogen cavitation and an OptiPrep gradient. The ER and plasma membrane derived vesicles exhibit different densities because they contain different phospholipids and associated proteins. ER originated vesicles are much denser than those obtained from the plasma membrane. The OptiPrep gradient was selected because of its superior ability to maintain isosmotic pressure independent of the density of the gradient used to isolate cellular organelles and subcellular vesicles (Graham et al., 1994). We applied this method to study ligand induced changes in the assembly of nicotinic receptors in the ER as well as the plasma membrane and validated our method with Western blotting and single molecule step-wise photobleaching. We believe this method can be applied for virtually any type of transmembrane proteins to conduct single molecule studies and to understand organelle-specific structural and functional properties.
Materials and Reagents
Cell culture flasks (Greiner Bio One International, catalog number: 658175 )
50 ml centrifuge tubes (VWR, catalog number: 89039-658 )
Ultra-Clear ultracentrifuge tubes (Beckman Coulter, catalog number: 344061 )
Four 5-ml Serological pipettes (VWR, catalog number: 89130-896 )
One 9-inch-flint-glass Pasteur pipette (VWR, catalog number: 14672-380 )
15 ml centrifuge tubes (VWR, catalog number: 89039-666 )
1.5-ml tubes
Wine cork
Razor blades (VWR, catalog number: 55411-050 )
Two ice buckets (VWR, catalog number: 10146-202 )
HEK293T cells (Sigma-Aldrich, catalog number: 85120602-1VL )
Deionized water
PBS buffer (VWR, catalog number: 97062-948 )
Versene solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15040066 )
Nitrogen gas
OptiPrep (60% solution, Sigma-Aldrich, catalog number: D1556-250ML )
Anti-calnexin (Abcam, catalog number: ab92573 )
Anti-sodium potassium ATPase (Abcam, catalog number: ab76020 )
Mouse anti-Rabbit secondary antibody (Santa Cruz Biotechnology, catalog number: sc-2357 )
ClarityTM Western ECL Substrate (Bio-Rad Laboratories, catalog number: 1705060 )
Tris-HCl (Sigma-Aldrich, Roche Diagnostics, catalog number: 10812846001 )
Sodium chloride (NaCl) (Fisher Scientific , catalog number: BP358-1 )
Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: BP214-500 )
Calcium chloride (CaCl2) (Fisher Scientific, catalog number: C614-500 )
Protease inhibitor mini tablet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: A32955 )
Sucrose (VWR, catalog number: 97063-788 )
HEPES (Fisher Scientific, catalog number: BP310-500 )
Hypotonic protease inhibitor solution (see Recipes)
Sucrose buffer (see Recipes)
Equipment
One 1 ml pipettor (Gilson, catalog number: F123602 )
One pipet controller (VWR, catalog number: 613-4442 )
One pair of scissors
A set of stand (a stand and a clamp)
Incubator
Biosafety cabinet (Class II, Type A2)
Centrifuge (Beckman Coulter, model: Allergra® X-22R ; Rotor: Beckman Coulter, model: SX4250 )
Ultracentrifuge (Beckman Coulter, model: L-60 )
Swing bucket rotor (Beckman Coulter, model: SW 28 )
Fixed angle rotor (Beckman Coulter, model: 70 Ti )
Belly dancer or orbital shaker
Nitrogen cavitation chamber/vessel(Parr Instrument Company, catalog number: 4639 )
Variable-Flow Peristaltic Pumps (Fisherbrand, Thermo Fisher)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Moonschi, F. H., Fox-Loe, A. M., Fu, X. and Richards, C. I. (2018). Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies. Bio-protocol 8(9): e2825. DOI: 10.21769/BioProtoc.2825.
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Category
Molecular Biology > Protein > Isolation
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2,826 | https://bio-protocol.org/exchange/protocoldetail?id=2826&type=0 | # Bio-Protocol Content
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Peer-reviewed
Glycogen and Extracellular Glucose Estimation from Cyanobacteria Synechocystis sp. PCC 6803
MK Md. Rezaul Islam Khan
YW Yushu Wang
SA Shajia Afrin
LH Lin He
GM Gang Ma
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2826 Views: 5907
Original Research Article:
The authors used this protocol in Sep 2016
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Sep 2016
Abstract
Cyanobacteria, which have the extraordinary ability to grow using sunlight and carbon dioxide, are emerging as a green host to produce value-added products. Exploitation of this highly promising host to make products may depend on the ability to modulate the glucose metabolic pathway; it is the key metabolic pathway that generates intermediates that feed many industrially important pathways. Thus, before cyanobacteria can be considered as a leading source to produce value-added products, we must understand the interaction between glucose metabolism and other important cellular activities such as photosynthesis and chlorophyll metabolism. Here we describe reproducible and reliable methods for measuring extracellular glucose and glycogen levels from cyanobacteria.
Keywords: Extracellular glucose Glycogen Cyanobacteria Synechocystis sp. PCC 6803
Background
Cyanobacteria have a light-dark cycle in their natural habitat. In the light, their metabolism is centered on photosynthesis, the Calvin cycle, glycolysis and the TCA cycle with N-assimilation; carbon is stored as glycogen. In the dark, glycogen is metabolized through glycolysis and the oxidative pentose phosphate (OPP) pathway, the oxidative and reductive branches of the TCA cycle, and the C4 cycle (Nagarajan et al., 2014). Thus, the shift from dark to light or light to dark drives metabolic reprogramming.
In the laboratory, the addition of glucose to the culture media also impacts cyanobacteria metabolic programs. For example, nutritional and environmental conditions influence how the cyanobacterium Synechocystis metabolizes glucose; Synechocystis metabolizes glucose differently in photoautotrophic, heterotrophic and mixotrophic conditions. Previous studies reported that some strains of Synechocystis are light-dependent and glucose tolerant (Anderson and McIntosh, 1991). Light-activated heterotrophic growth (LAHG) conditions are characterized by the presence of glucose and growth in the dark with a pulse of white or blue light for at least 5-15 min per day. However, some strains of Synechocystis are glucose intolerant, meaning that they cannot grow in the presence of glucose in the dark. In summary, the addition of glucose to the culture media of Synechocystis has been reported to bring physiological and metabolic changes such as pigmentation (Ryu et al., 2004), carbon metabolism (Lee et al., 2007; Takahashi et al., 2008), phosphorylation patterns (Bloye et al., 1992), carbon dioxide uptake (Kaplan and Reinhold, 1999), and oxidative stress generation (Narainsamy et al., 2013).
To identify the utility of cyanobacteria to produce natural product, growing cyanobacteria in large-scale is a prerequisite. For growing cyanobacteria efficiently, it’s important to characterize the direct impact of common environmental factors such as light and temperature on glucose metabolism. Here, we present an accurate, reproducible, and reliable method to quantify extracellular glucose and glycogen levels of cyanobacteria, we belelive that this method will help determine the utility of cyanobacteria as a source for engineering natural products.
Materials and Reagents
Pipette tips (20 µl-1 ml, autoclaved)
Aluminum foil
1.5 and 2 ml Eppendorf tubes (autoclaved)
0.45 µm filter
Cyanobacteria Synechocystis sp. PCC 6803 (WT, mutant D95 & C95)
Note: For more information about these strains, please see Data analysis A4.
Sulfuric acid (ACS reagent, Sigma-Aldrich, catalog number: S1526 )
Nitrogen
Ethanol (Sigma-Aldrich, catalog number: 362808-1L )
Glycogen (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0561 )
Amyloglucosidase (Sigma-Aldrich, catalog number: 10115-1G-F )
Sodium carbonate (Na2CO3) (Sangon Biotech, catalog number: ST0840 )
Sodium nitrate (NaNO3) (Sinopharm Chemical Reagent, catalog number: 10019918 )
Hydrochloric acid (HCl) (Sinopharm Chemical Reagent, catalog number: 10011018 )
Sodium hydroxide (NaOH) (Sangon Biotech, catalog number: SB0617 )
Potassium phosphate dibasic (K2HPO4) (Sangon Biotech, catalog number: PB0447 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sangon Biotech, catalog number: MT0864 )
Ferric ammonium sulphate (Sangon Biotech, catalog number: A502657 )
Citric acid (Sangon Biotech, catalog number: C0529 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sangon Biotech, catalog number: CT1331 )
EDTA-Na2 (Sangon Biotech, catalog number: E0105 )
Boric acid (H3BO3) (Sangon Biotech, catalog number: BB0044 )
Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sangon Biotech, catalog number: A500331 )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sangon Biotech, catalog number: A602906 )
Sodium molybdate dehydrate (Na2MoO4·2H2O) (Sangon Biotech, catalog number: SB0865 )
Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sangon Biotech, catalog number: A501425 )
Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Sangon Biotech, catalog number: CB7774 )
Sodium thiosulfate anhydrous (Na2S2O3) (Sangon Biotech, catalog number: S1712 )
D-glucose (Sangon Biotech, catalog number: 501991 )
Glucose standard solution (Sigma-Aldrich, catalog number: G3285 )
Benzoic acid (Sinopharm Chemical Reagent, catalog number: 30018615 )
Glucose oxidase/peroxidase (Sigma-Aldrich, catalog number: G3660 )
o-Dianisidine reagent (Sigma-Aldrich, catalog number: D2679 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P6310 )
Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Sangon Biotech, catalog number: TB0927 )
BG-11 media (see Recipes)
D-Glucose (see Recipes)
Assay reagent (see Recipes)
30% (w/v) KOH (see Recipes)
100 mM sodium acetate (pH 4.5) (see Recipes)
Equipment
Conical flasks (100, 200, 500 ml) (SHUNIU, Chengdu, China)
Vortex (FINE PCR, model: Finevortex )
Centrifuge machine, unrefrigerated, maximum speed 17,000 x g, with rotor for microtubes (Thermo Fisher Scientific, model: HeraeuesTM PicoTM 17 )
Glass test tubes 18 x 150 mm
Spectrophotometer (METASH, model: V-5600 )
1 ml glass cuvettes (METASH)
Pipettes (20 µl, 200 µl, 1 ml, 5 ml) (Gilson, France)
Water baths (temperature set at 37 ± 1 °C; 60 °C; 92 °C) (Meier, model: XMTD-204 )
Shaking light-incubator (light:up to 150 µmoles/m2/sec; temperature: 20-50 °C) (Shanghai Zhichu, model: ZQWY-200G )
Freeze dryer (Labconco, model: FreeZone Plus 6 )
Oven, 60 °C (Boxun, model: GZX-9140MBE )
Autoclave (Boxun, model: YXQ-LS-100SII )
pH meter (Mettler Toledo, model: FE 20 )
Swimming holders
Software
GraphPad PRISM (Version 5.01)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Khan, M. R. I., Wang, Y., Afrin, S., He, L. and Ma, G. (2018). Glycogen and Extracellular Glucose Estimation from Cyanobacteria Synechocystis sp. PCC 6803. Bio-protocol 8(9): e2826. DOI: 10.21769/BioProtoc.2826.
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Category
Microbiology > Microbial biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Glycogen
Biochemistry > Carbohydrate > Polysaccharide
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2,827 | https://bio-protocol.org/exchange/protocoldetail?id=2827&type=0 | # Bio-Protocol Content
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Peer-reviewed
Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining
HO Hiroyuki Ohsaki
TM Toru Matsunaga
TF Taishi Fujita
YT Yasunori Tokuhara
SK Shingo Kamoshida
TS Tadashi Sofue
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2827 Views: 6564
Reviewed by: Di WangHaixia Xu
Original Research Article:
The authors used this protocol in Dec 2017
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Dec 2017
Abstract
In glomerular disease, podocytes and parietal epithelial cells (PECs) are shed in the urine. Therefore, urinary podocytes and PECs are noninvasive biomarkers of glomerular disease. The purpose of this protocol is to employ immunocytochemistry to detect podocytes and PECs, using the WT1 antibody on liquid-based cytology slides.
Keywords: Podocytes Parietal epithelial cells Liquid-based cytology WT1 Immunocytochemistry Glomerular disease Crescent formation Urine
Background
Podocytes line the exterior of glomerular capillaries and thus face the Bowman’s capsule and primary urine. In glomerular injury, podocytes may detach from the glomerular basement membrane. The detachment of podocytes and their shedding in the urine have been implicated in the progression of glomerular diseases and crescent formation. Parietal epithelial cells (PECs) cover the inner aspect of the Bowman’s capsule. Similar to podocytes, it has been reported that PECs positive for WT1, proliferate and are shed in the urine during active glomerular disease (Zhang et al., 2012; Fujita et al., 2017).
Previous studies have shown a relationship between the number of urinary podocytes and PECs and various types of glomerular disease (Hara et al., 1998; Nakamura et al., 2000; Achenbach et al., 2008). The studies cited above used conventional methods (direct smears and cytospin) to prepare the urine samples. However, these conventional methods have common problems, including an air-drying effect and the possibility of significant cell loss during the cytocentrifugation and fixation processes. In addition, standardization and quantitative evaluation are difficult with the conventional methods. Although most previous studies have used immunofluorescence staining with the podocalyxin antibody, this method is difficult to perform in small- and medium-sized hospitals because of the specialty antigen and the requirement for a fluorescence microscope.
A useful urinary biomarker test should be easy to perform and have minimal requirements for standardized sample preparation and immunocytochemistry. Therefore, we have devised a method for detecting podocytes and PECs by combining liquid-based cytology, the WT1 antibody, and immunoenzyme staining. SurePathTM liquid-based cytology was developed as a replacement for the conventional sample preparation method because of its better cell preservation and higher cell recovery rate. SurePathTM allows the possibility of standardization and quantitative evaluation, and this system can be operated manually without machines. Furthermore, immunoenzyme staining using the WT1 antibody is performed in most pathological laboratories. Therefore, our method can be an inexpensive, simple, and internationally standardized method for the detection of urinary podocytes and PECs.
Materials and Reagents
Saline-soaked gauze balls
Medicine spoon
Conical centrifuge tube (Corning, Falcon®, catalog number: 352097 ) or equivalent
Kimwipes (KCWW, Kimberly-Clark, catalog number: 34133 ) or equivalent
PAP pen (Abcam, catalog number: ab2601 ) or equivalent
BD TotalysTM Slide Prep Slide Rack (BD, catalog number: 491294 )
Coverslip (Matsunami Glass, catalog number: C024321 ) or equivalent
BD SurePathTM Manual Method Kit (including settling chamber and positively charged slide) (BD, catalog number: 491266 )
BD CytoRichTM Red Preservative Fluid (BD, catalog number: 491336 )
Ethanol (Wako Pure Chemical Industries, catalog number: 059-06957 ) or equivalent
Methanol (Wako Pure Chemical Industries, catalog number: 136-09475 ) or equivalent
Hydrogen peroxide (Wako Pure Chemical Industries, catalog number: 086-07445 ) or equivalent
Phosphate-buffered saline (PBS) (Nichirei Biosciences, catalog number: 415223 ) or equivalent
WT1 antibody (clone 6F-H2) (Agilent Technologies, catalog number: M3561 )
Antibody diluent (Agilent Technologies, catalog number: S3022 ) or equivalent
N-Histofine® Simple StainTM MAX PO (MULTI) (Nichirei Biosciences, catalog number: 414151F )
N-Histofine® DAB-3S Kit (Nichirei Biosciences, catalog number: 415192F )
Mayer’s hematoxylin (Muto Pure Chemicals, catalog number: 30002 ) or equivalent
Xylene (Wako Pure Chemical Industries, catalog number: 245-00717 ) or equivalent
Malinol (mounting medium) (Muto Pure Chemicals, catalog number: 20093 ) or equivalent
Equipment
Pipette (Nichiryo, model: Nichipet EXII ) or equivalent
Centrifuge (Kubota, model: M4000 ) or equivalent
Vortex mixer (Scientific Industries, model: Vortex-Genie 2 ) or equivalent
Microscope (Olympus, model: BX43 ) or equivalent
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ohsaki, H., Matsunaga, T., Fujita, T., Tokuhara, Y., Kamoshida, S. and Sofue, T. (2018). Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining. Bio-protocol 8(9): e2827. DOI: 10.21769/BioProtoc.2827.
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Category
Cell Biology > Cell staining > Protein
Cell Biology > Cell-based analysis > Immunocytochemistry
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2,828 | https://bio-protocol.org/exchange/protocoldetail?id=2828&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Modified Low-quantity RNA-Seq Method for Microbial Community and Diversity Analysis Using Small Subunit Ribosomal RNA
YY Yong-Wei Yan
TZ Ting Zhu
BZ Bin Zou
Zhe-Xue Quan
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2828 Views: 4994
Edited by: Dennis Nürnberg
Reviewed by: Stefan de VriesShyam Solanki
Original Research Article:
The authors used this protocol in Oct 2017
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Abstract
We propose a modified RNA-Seq method for small subunit ribosomal RNA (SSU rRNA)-based microbial community analysis that depends on the direct ligation of a 5’ adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10-100 ng) and does not require a DNA removal step. Using this method, we could obtain more 16S rRNA sequences of the same regions (variable regions V1-V2) without the interference of DNA in order to analyze OTU (operational taxonomic unit)-based microbial communities and diversity. The generated SSU rRNA sequences are also suitable for the coverage evaluation for bacterial universal primer 8F (Escherichia coli position 8 to 27), which is commonly used for bacterial 16S rRNA gene amplification. The modified RNA-Seq method will be useful to determine potentially active microbial community structures and diversity for various environmental samples, and will also be useful for identifying novel microbial taxa.
Keywords: RNA-Seq Low quantity SSU rRNA OTU Microbial community
Background
Ribosomal RNA (rRNA) accounts for more than 90% of the total microbial RNA, and is suitable for the analysis of microbial communities as an indicator of microbial physiological activity to synthesize proteins (Blazewicz et al., 2013). The study of microbial community transcripts, including rRNA and mRNA, in a particular environment (Double RNA metatranscriptomics) has advantages in providing both functional and taxonomic information on microbes (Urich et al., 2008), but has failed to perform OTU-based community comparisons. Although diversity indices could be calculated and compared using the V3 region of 16S rRNA sequences when gel-extracted SSU rRNA is analyzed, only a third of the resulting 16S rRNA sequences were found to be suitable for such analyses (Li et al., 2014). Besides, such method usually requires high quantities of RNA (Li et al., 2014). We recently developed a modified RNA-Seq method that uses an immediate adaptor ligation step at the 5’ end of the RNA prior to reverse transcription. Consequently, we can obtain more 16S RNA reads which can be used for OTU-based community and diversity analysis especially regarding low-RNA-yield samples such as tap water, shower curtain and human skin (Yan et al., 2017), and also mudflat sediment samples (Yan et al., 2018).
Materials and Reagents
RNA extraction
Glass beads, acid-washed (≤ 106 μm) (Sigma-Aldrich, catalog number: G4649 )
2 ml bead beating tubes (QIAGEN, catalog number: 13118-400 )
mirVANATM miRNA Isolation Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1560 )
RNA-Seq library preparation
PCR tubes (Corning, Axygen®, catalog number: PCR-02-L-C )
RNA-Seq Library Preparation Kit for Whole Transcriptome Discovery–Illumina Compatible (Gnomegen, catalog number: K02421-T )
DNase/RNase free water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9932 )
QubitTM RNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32855 )
RNaseOUTTM Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777019 )
QubitTM dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
Gnome Size Selector (Gnomegen, catalog number: R02424S )
Other materials
Pipette tips (10 μl, 200 μl, 1,000 μl) (Corning, Axygen®, catalog numbers: TF-300-R-S , TF-200-R-S , TF-1000-R-S )
1.5 ml Eppendorf tubes (Eppendorf, catalog number: 022363204 )
100% ethanol
RNase-free 70% ethanol
Agarose (Biowest, catalog number: 111860 )
50x TAE Buffer (Sangon Biotech, catalog number: B548101-0500 )
DL2,000 DNA Marker (Takara Bio, catalog number: 3427A )
Equipment
Pipettes (0-10 μl, 10-100 μl, 100-1,000 μl) (Eppendorf, catalog numbers: k03030 , k03031 , k03032 )
Vortex-genieTM 2 (QIAGEN, model: Vortex-Genie® 2 )
Fresco 17 centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM FrescoTM 17 )
Qubit 2.0 Fluorometer (Thermo Fisher Scientific, model: Qubit 2.0 )
Automated thermal cycler TP600 (Takara Bio, model: TP600 )
MagneSphere® Technology Magnetic Separation Stand (twelve-position) (Promega, catalog number: Z5342 )
Electrophoresis apparatus (Bio-Rad Laboratories)
Gel imaging system
Software
Sickle v1.33 (Joshi and Fass, 2011)
Mothur v1.33.3 (Schloss et al., 2009)
QIIME v1.8.0 (Caporaso et al., 2010)
MIPE (Zou et al., 2017)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Yan, Y., Zhu, T., Zou, B. and Quan, Z. (2018). A Modified Low-quantity RNA-Seq Method for Microbial Community and Diversity Analysis Using Small Subunit Ribosomal RNA. Bio-protocol 8(9): e2828. DOI: 10.21769/BioProtoc.2828.
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Category
Microbiology > Community analysis > RNA-Seq
Molecular Biology > RNA > RNA sequencing
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2,829 | https://bio-protocol.org/exchange/protocoldetail?id=2829&type=0 | # Bio-Protocol Content
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Purification of Total RNA from DSS-treated Murine Tissue via Lithium Chloride Precipitation
Emilie Viennois
AT Anika Tahsin
DM Didier Merlin
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2829 Views: 8051
Edited by: Andrea Puhar
Reviewed by: Bruno LamasSaskia F. Erttmann
Original Research Article:
The authors used this protocol in Sep 2013
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Sep 2013
Abstract
We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.
Keywords: Dextran sodium sulfate Colitis Lithium chloride RNA q-RT-PCR
Background
Dextran Sulfate Sodium (DSS) is very commonly used in laboratories to induce colitis in rodents. Specifically, it mimics the clinical and histological features of human Inflammatory Bowel Disease (IBD) with Ulcerative Colitis (UC) characteristics. DSS is diluted in the drinking water and penetrates tissues. We have observed that contamination of RNA extracts with DSS prevented successful subsequent amplification processes from the colon and small intestine, but also blood and other tissues obtained from DSS-treated animals. We had previously shown that the presence of DSS in the samples inhibited reverse transcription and polymerase chain reaction amplification (Viennois et al., 2013). This inhibitory effect was observed in a dose depended manner by Kerr et al. and they suggested a poly-A-purification based technique to remove DSS from total RNA extract (Kerr et al., 2012). We hereby propose another efficient and economical method for purifying total RNA extracts from DSS traces based on lithium chloride (LiCl) precipitations. This method has been extensively used in our laboratory as well as in others (Chassaing et al., 2012, Li et al., 2016); however, no attempt has been taken to document the procedure in detail. Therefore, we provide a detailed description of LiCl purification procedure of total RNA primarily isolated from DSS-treated murine tissue with another method (Trizol, Spin column-based nucleic acid purification…).
Materials and Reagents
Pipette tips (0.1-10 µl, 1-200 µl, 100-1,000 µl)
Eppendorf Safe-Lock Tubes, 1.5 ml (Eppendorf, catalog number: 022363204 )
8 M lithium chloride (LiCl) (SIGMA Lithium Chloride Solution, 8 M Solution, Sigma-Aldrich, catalog number: L7026-100ML , 090M8728)
RT-PCR Grade Water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9935 )
Pure 100% ethanol (Decon Labs, catalog number: 2716 )
3 M sodium acetate, pH 5.2 (see Recipes)
70% ethanol (see Recipes)
Equipment
Pipettes 0.5-10 µl, 10-100 µl and 100-1,000 µl (Eppendorf, model: Research® plus , Variable Adjustable Volume Pipettes)
Refrigerated centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM Micro 21R , or equivalent)
Multi-Mode Microplate Reader (BioTek Instruments, model: BioTekTM SynergyTM 2 )
-20 °C freezer
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Viennois, E., Tahsin, A. and Merlin, D. (2018). Purification of Total RNA from DSS-treated Murine Tissue via Lithium Chloride Precipitation. Bio-protocol 8(9): e2829. DOI: 10.21769/BioProtoc.2829.
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Category
Immunology > Inflammatory disorder
Immunology > Mucosal immunology > Digestive tract
Molecular Biology > RNA > qRT-PCR
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283 | https://bio-protocol.org/exchange/protocoldetail?id=283&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analyze p53 degradation by 35S p53 Pulse Chase Analysis
MA Megan Astle
RP Richard Pearson
KH Katherine Hannan
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.283 Views: 9474
Original Research Article:
The authors used this protocol in Apr 2012
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Abstract
p53, is known as the guardian of the genome and as such requires exquisite regulation not only of its abundance but also its activity. The abundance of p53 can be modulated at the level of transcription, translation, and also via its degradation.
This protocol involves 35S metabolic labelling of newly synthesized proteins followed by a period of chase with "cold" media. Samples are harvested and p53 immunopreciptiated, separated by SDS PAGE and the levels of 35S labelled p53 determined. By comparing the level of 35S p53 at 0 h to those "chased" with cold media (e.g. 60 min) provides an indication of the rate of p53 turnover.
Materials and Reagents
Dulbecco’s Modified Eagle Medium (DMEM) methionine and cysteine free media (1x) (Life Technologies, Invitrogen™, catalog number: 21013-024 )
Dialysed fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 26400-036 )
Express 35S protein labelling mix (PerkinElmer, catalog number: NEG 0720008MC )
Cell lifter (Corning Incorporated, catalog number: 3008 )
Protein A sepharose (Zymed, catalog number: 10-1042 )
p53 DO-1 (Santa Cruz, catalog number: SC-126 )
DC Protein assay kit (Bio-Rad Laboratories, catalog number: 500-0116 )
Bio-Rad safe stain solution (Bio-Rad Laboratories, catalog number: 161-0786 )
L-glutatmine or Glutamax (Life Technologies, Invitrogen™, catalog number: 35050-061 )
cOmplete Ultra tablets (Roche Applied Science, catalog number: 05 892 988 001 )
PhosSTOP tablets (Roche Applied Science, catalog number: 049068450001 )
NP-40 (IGEPAL, catalog number: CA-630 )
Beta mercapotethanol
Bromophenol blue
Complete media (see Recipes)
Starvation media (see Recipes)
Dulbecco’s PBS (see Recipes)
1x lysis buffer (see Recipes)
2x SLB buffer (see Recipes)
Washed protein A sepharose (see Recipes)
Equipment
Bioruptor sonicator (Diagenode)
Macsmix tube rotator (Miltenyi Biotech GmbH) or standard Rotating wheel
Heat block
SDS–polyacrylamide gel electrophoresis (PAGE) running equipment
Orbital shaker
3 mm whatman paper and plastic wrap
Gel Dryer and pump
Storage phosphor screen (Molecular Dynamics) and Typhoon Trio (GE Healthcare)
Software
Image Quant software (Molecular Dynamics)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Astle, M., Pearson, R. and Hannan, K. (2012). Analyze p53 degradation by 35S p53 Pulse Chase Analysis. Bio-protocol 2(21): e283. DOI: 10.21769/BioProtoc.283.
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Category
Biochemistry > Protein > Immunodetection
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2,830 | https://bio-protocol.org/exchange/protocoldetail?id=2830&type=0 | # Bio-Protocol Content
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Time-of-addition and Temperature-shift Assays to Determine Particular Step(s) in the Viral Life Cycle that is Blocked by Antiviral Substance(s)
CA Chie Aoki-Utsubo*
MC Ming Chen*
Hak Hotta
*Contributed equally to this work
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2830 Views: 8483
Edited by: Vamseedhar Rayaprolu
Reviewed by: Kristin ShinglerSmita Nair
Original Research Article:
The authors used this protocol in Nov 2017
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Abstract
Viruses infect their host cells to produce progeny virus particles through the sequential steps of the viral life cycle, such as viral attachment, entry, penetration and post-entry events. This protocol describes time-of-addition and temperature-shift assays that are employed to explore which step(s) in the viral life cycle is blocked by an antiviral substance(s).
Keywords: Viral life cycle Time-of-addition assay Pretreatment Co-treatment Post-entry treatment Temperature-shift assay Attachment Penetration
Background
Viruses are obligate intracellular parasites that hijack host cell machineries to replicate their own genome. The viral life cycle proceeds through the attachment (binding) of an infectious viral particle (virion) to the surface of the host cell and its penetration (internalization, fusion) into intracellular compartments, where virion uncoating (disassembly) takes place, followed by viral genome transcription/replication, viral protein synthesis and virion assembly, which eventually results in the production and release of progeny virions from the infected cell (Scheel and Rice, 2013).
To explore which step(s) of the viral lifecycle is blocked by an antiviral substance, time-of-drug addition experiments are performed. In brief, an antiviral substance is added to the virus and/or host cells at different time points relative to viral inoculation to the cells (Chen et al., 2017): (1) Pre-treatment of the cells with an antiviral substance before viral inoculation examines whether the substance could block the viral receptor to inhibit viral attachment to the host cells or if it could induce production of antiviral host factors, such as interferon. (2) Pre-treatment of virus followed by inoculation of the treated virus to the cells examines the virucidal or neutralizing activity of the antiviral substance. (3) Co-treatment of cells and virus during virus inoculation examines the antiviral effect on the virus entry steps including virucidal (neutralizing) activity and blockade of viral attachment and penetration to the cells. (4) Treatment of virus-infected cells during the entire post-inoculation period examines the antiviral effect during the post-entry steps, such as genome translation and replication, virion assembly and virion release from the cells. In addition, temperature-shift assay can differentiate between (5) antiviral activity against attachment that occurs at both 37 °C and 4 °C and (6) antiviral activity against penetration (internalization and/or fusion) that occurs only at 37 °C. An interesting example is that secreted phospholipase A2 obtained from bee venom inhibits penetration of human immunodeficiency virus (HIV) virion without inhibiting virion attachment to the cell surface (Fenard et al., 1999).
In this article, we describe procedures of time-of-addition and temperature-shift assays for the mechanistic analyses of antiviral substances using a fluorescent antibody (FA) method, which have been reported elsewhere (Wahyuni et al., 2013; Adianti et al., 2014; Ratnoglik et al., 2014; El-Bitar et al., 2015; Apriyanto et al., 2016; Chen et al., 2017). Alternative titration methods, such as plaque assay, 50% tissue culture infectious dose (TCID50) assay and quantitative PCR (qPCR and qRT-PCR), are also used to determine viral titers as described elsewhere.
Materials and Reagents
Disposable tips
10 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3510-05 )
200 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3900 )
1 ml capacity (FUKAEKASEI and WATSON, catalog number: 110-502C )
100 mm culture dish (Corning, Falcon®, catalog number: 353003 )
24-well culture plate (Corning, Falcon®, catalog number: 353047 )
Cover slip (13 mm in diameter; Matsunami Glass, catalog number: C013001 )
1.5 ml Microcentrifuge tube (FUKAEKASEI and WATSON, catalog number: 131-715C )
Disposable serological pipette
1 ml capacity (Corning, Falcon®, catalog number: 356521 )
5 ml capacity (Iwaki, catalog number: 7153-005 )
10 ml capacity (Iwaki, catalog number: 7154-010 )
Huh7it-1 cells (Apriyanto et al., 2016)
Viruses (Chen et al., 2017):
Hepatitis C virus (HCV; J6/JFH-1 strain)
Dengue virus type 2 (DENV-2; Trinidad 1751 strain)
Japanese encephalitis virus (JEV; Nakayama strain)
Influenza A virus (FLUAV; A/Udorn/307/72[H3N2])
Sendai virus (SeV; Fushimi strain)
Herpes simplex virus type 1 (HSV-1; CHR3 strain)
Coxsackievirus B3 (CV-B3; Nancy strain)
Crushed ice
4% paraformaldehyde (Wako Pure Chemical Industries, catalog number: 163-20145 )
Triton X-100 (Wako Pure Chemical Industries, catalog number: 169-21105 )
Bovine serum albumin (BSA; Wako Pure Chemical Industries, catalog number: 015-21274 )
Primary antibodies (Chen et al., 2017): Antibodies against viruses, such as HCV, DENV-2, JEV, FLUAV, SeV, HSV-1 and CV-B3
Secondary antibodies:
FITC-conjugated goat anti-human IgG (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 104AG )
Alexa Flour488-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Invitrogen, catalog number: A-11001 )
Alexa Flour488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Invitrogen, catalog number: A-11008 )
Hoechst 33342 (Thermo Fisher Scientific, catalog number: H3570 )
Vectashield solution (Vector Laboratories, catalog number: H-1000 )
Trypsin-EDTA solution (Wako Pure Chemical Industries, catalog number: 209-16941 )
High glucose Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries, catalog number: 044-29765 )
MEM with non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 10370-021 )
Fetal bovine serum (FBS; Biowest, catalog number: S1820 )
Penicillin-Streptomycin solution (Wako Pure Chemical Industries, catalog number: 168-23191 )
Sodium chloride (NaCl; Wako Pure Chemical Industries, catalog number: 191-01665 )
Potassium chloride (KCl; Wako Pure Chemical Industries, catalog number: 163-03545 )
Sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O; Sigma-Aldrich, catalog number: 71649 )
Potassium dihydrigen phosphate (KH2PO4; Wako Pure Chemical Industries, catalog number: 169-04245 )
Sodium citrate dehydrate (Sigma-Aldrich, catalog number: W302600 )
Citric acid (Wako Pure Chemical Industries, catalog number: 030-05525 )
Culture medium (see Recipes)
10x phosphate-buffered saline without Ca2+ and Mg2+ (PBS[-]) and 1x PBS(-) (see Recipes)
Citrate buffer (see Recipes)
Equipment
Biosafety cabinet (e.g., Panasonic, model: MHE-S1301A2 )
CO2 incubator (e.g., Panasonic, model: MCO-20AIC )
Autoclave (e.g., TOMY SEIKO, model: SX-500 )
Refrigerated tabletop centrifuge (e.g., Eppendorf, model: 5424 )
Micropipette (Gilson, P20, P200, P1000)
Hemocytometer (e.g., Erma, catalog number: 03-303-1 )
-80 °C freezer (e.g., PHC, model: MDF-384 )
Inverted microscope (e.g., Olympus, model: CKX53 )
Fluorescent microscope (e.g., Carl Zeiss, model: LSM700 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Aoki-Utsubo, C., Chen, M. and Hotta, H. (2018). Time-of-addition and Temperature-shift Assays to Determine Particular Step(s) in the Viral Life Cycle that is Blocked by Antiviral Substance(s). Bio-protocol 8(9): e2830. DOI: 10.21769/BioProtoc.2830.
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Category
Microbiology > Antimicrobial assay > Antiviral assay
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2,831 | https://bio-protocol.org/exchange/protocoldetail?id=2831&type=0 | # Bio-Protocol Content
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Assessing the Efficacy of Small Molecule Inhibitors in a Mouse Model of Persistent Norovirus Infection
Jana Van Dycke
Johan Neyts
JR Joana Rocha-Pereira
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2831 Views: 5568
Edited by: Yannick Debing
Reviewed by: Kristin ShinglerBalaji Olety Amaranath
Original Research Article:
The authors used this protocol in Mar 2016
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Mar 2016
Abstract
Human norovirus is the most common cause of acute gastroenteritis worldwide, resulting in estimated mortality of ~210,000 each year, of whom most are children under the age of five. However, norovirus can infect people of all age groups. There is a risk of prolonged infection in children, the elderly and patients who are immunocompromised. To study the inhibition of persistent norovirus replication by small molecule antivirals in vivo, we used a murine norovirus CR6 strain (MNV.CR6). We demonstrated earlier that efficient small molecules can reduce viral shedding in the stool of infected mice. Here we present how to generate the MNV.CR6 virus stock, infect type I and II interferon receptor knockout AG129 mice via oral gavage, administer antivirals to mice, and quantify viral genome copies in the stool of these mice.
Keywords: Murine norovirus Persistent infection Oral gavage RT-qPCR Antiviral
Background
Human noroviruses are an important cause of gastroenteritis. Although most norovirus infections are acute and self-limiting, the infection can become chronic in patients with an immunodeficient status, particularly in solid organ and hematopoietic stem cell transplant recipients, patients undergoing chemotherapy and patients with AIDS (Westhoff et al., 2009; Green, 2014; Angarone et al., 2016). Prolonged norovirus infection is also observed in young children and the elderly resulting in an increased duration of illness, increased defecations and virus shedding for up to 47 days (Murata et al., 2007; Aoki et al., 2010). Reduction of immunosuppressive therapy is, when feasible, the strategy of choice to control the infection in transplant recipients. Specific antiviral therapies to treat (chronic) norovirus gastroenteritis are not available. To assess the inhibitory effect of small molecules on persistent norovirus infections, we used a mouse-adapted persistent murine norovirus (MNV.CR6) in type I and II interferon receptor knockout AG129 mice (Strong et al., 2012). MNV is a genogroup V norovirus that is widely used as a surrogate for human noroviruses, which comprises around 30 strains (Karst et al., 2003; Wobus et al., 2004). The MNV.CR6 is avirulent in AG129 and STAT1-/- mice, but replicates for weeks to months in wild type mice and to higher titers in innate immune-deficient mice (Thackray et al., 2007). The MNV.CR6 strain has a tropism for the proximal colon and the cecum, where it persists and replicates more efficiently than the MNV.CW3 strain, which causes acute infection (Arias et al., 2012; Nice et al., 2013).
Materials and Reagents
Overall
Appropriate personal protection to work in a biosafety level 2 (BSL-2) laboratory or A-2 animal facility (gloves, lab coat, hairnet, shoe covers, safety glasses)
Disinfectant: bleach (5,000 ppm) or Virkon S.
In vitro work
Culture flasks (150 cm2, TPP, catalog number: 90856 )
Cell scrapers (Corning, Falcon®, catalog number: 353086 )
Cryotubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 377224 )
Pipet tips (10 µl, 100 µl, 1,000 µl)
Disposable serological pipets (5 ml, 10 ml, 25 ml)
Murine macrophage cells (RAW 264.7, ATCC, catalog number: TIB-71 )
Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, catalog number: 14190094 )
Dulbecco’s modified eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 41965039 )
Fetal calf serum (FCS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
Sodium bicarbonate (Thermo Fisher Scientific, GibcoTM, catalog number: 25080060 )
L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030024 )
HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630056 )
Penicillin/streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360039 )
Culture medium (see Recipes)
In vivo work
Eppendorf safe-lock tubes, 1.5 ml (Eppendorf, catalog number: 0030120086 )
Syringe + needle for subcutaneous injection (VWR, catalog number: BDAM303176 )
Needle container (Sharpsafe, catalog number: 41602432 )
Bench surface protector (VWR, catalog number: 115-9220 )
Earmarks (Bioseb, catalog number: EP-1005-1 )
Plastic feeding tubes, 20 G x 30 mm, sterile (Instech Laboratories, catalog number: FTP-20-30 )
Sterile 1 ml syringe that fits on the feeding tube (VWR, catalog number: 612-0106 )
Plastic container to temporarily restrain a mouse
Ear tags (Bioseb, catalog number: EP-1005-1 ) + applicator (Style 1005s1)
Type I and II interferon receptor knockout AG129 mice (129/Sv mice), from BK Universal, UKCR6 strain
2’-C-methylcytidine (2CMC, Carbosynth, catalog number: NM07918 )
Favipiravir (T-705, BOC Sciences, catalog number: B0084-463609 )
Carboxymethylcellulose (CMC, Sigma–Aldrich, catalog number: C9481 )
Sterile saline (NaCl 0.9%, B. Braun)
Viral RNA quantification
Multipette tip of 0.2 ml (Eppendorf, catalog number: 0030089413 )
Lightcycler 480–Multiwell plate 96 (Roche Molecular Systems, catalog number: 04729692001 )
RNeasy Mini kit (QIAGEN, catalog number: 74106 )
Ethanol, absolute (Fisher Scientific, catalog number: 10342652 )
iTaq Universal Probes One-Step Kit (Bio-Rad Laboratories, catalog number: 1725141 )
Primers, probe and standard (Baert et al., 2008)
Equipment
Biosafety hood in an A-2 animal facility
Biosafety hood in a BSL-2 laboratory
Incubator (37 °C, 5% CO2, humidified)
Scale to weigh mice
Forceps suitable for picking up mouse feces (autoclavable)
Pipet set (P10, P100, P1000)
Pipetboy (Integra Biosciences, catalog number: 155016 )
Multipette® M4 (Eppendorf, catalog number: 4982000012 )
Multichannel pipette (Eppendorf, catalog number: 3122000035 )
-80 °C freezer
PCR Workstation
Vortex
Centrifuge with a rotor suitable for 1.5 ml tubes
Centrifuge with a rotor suitable for 50 ml tubes
Autoclave
RT-qPCR machine (Roche Diagnostics, model: Roche LightCycler® 96 )
Inverted light microscope
Cell counter
Software
GraphPad Prism Software, version 7
LightCycler® 96 SW 1.1 Software
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Dycke, J. V., Neyts, J. and Rocha-Pereira, J. (2018). Assessing the Efficacy of Small Molecule Inhibitors in a Mouse Model of Persistent Norovirus Infection. Bio-protocol 8(9): e2831. DOI: 10.21769/BioProtoc.2831.
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Category
Microbiology > in vivo model > Viruses
Molecular Biology > RNA > qRT-PCR
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2,832 | https://bio-protocol.org/exchange/protocoldetail?id=2832&type=1 | # Bio-Protocol Content
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Peer-reviewed
Growing and Pollinating Maize
SH Sarah Hake
China Lunde
Published: May 5, 2018
DOI: 10.21769/BioProtoc.2832 Views: 5125
Reviewed by: Avinash Chandra Pandey
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Abstract
Corn, also known as maize, has been a model organism for genetics since the 1900’s due to the ease of pollinations, large chromosomes and the prominent kernel and leaf traits with simple inheritance (Candela and Hake, 2008). It requires considerable water, light and fertilizer to reach reproductive maturity and can be best grown in the field or greenhouse. The following protocol outlines growing procedures and how to make controlled pollinations.
Keywords: Maize Controlled-pollinations Reproduction Plant biology
Materials and Reagents
1.5 ml centrifuge tubes
Soil for large pots (Multipurpose Blend)
Soil for trays and peet pots (Supersoil® Potting Soil)
Shoot bags: ‘Lawson 217’ 5000 bags/box
Tassel bags: ‘Lawson 402’ 1000 bags/box
Note: Colored bags are useful in a field with few pollinations.
Slow release fertilizer (Osmocote 14N-14P-14K; A90550)
Gnatrol (for insect control, available from Valent; EPA Reg. No. 73049-56)
Note: It must be used in compliance with Worker Protection Standard, 40 CFR Part 170.
Field stakes (Hummert International)
Row cover (Agribon)
Staples
Paperclips
Equipment
Planter (Hand Jab Slim-Style Planter)
11.35 L (3-gallon) pots (such as 3 gal pot from Viagrow available at Amazon.com)
Planting Trays (such as F1721 from T.O. Plastics available at Hummert International)
Plastic mulch for the field and drip system (Hummert International)
Drying oven set to 38 °C (such as those manufactured by Despatch)
Walk-in cooler with low humidity (such as those manufactured by Norlake)
Procedure
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Category
Plant Science > Plant physiology > Plant growth
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > Genotyping
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2,833 | https://bio-protocol.org/exchange/protocoldetail?id=2833&type=0 | # Bio-Protocol Content
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Infection Process Observation of Magnaporthe oryzae on Barley Leaves
Xiao-Lin Chen
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2833 Views: 8325
Edited by: Zhibing Lai
Reviewed by: Jun YangWende Liu
Original Research Article:
The authors used this protocol in Mar 2014
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The authors used this protocol in:
Mar 2014
Abstract
Rice blast and wheat blast caused by Magnaporthe oryzae is a serious threat to rice and wheat production. Appropriate methods for observing M. oryzae infection process are important for study of the fungal infection mechanisms, plant resistance reactions, and host-M. oryzae interactions. The rice leaf sheath is commonly used to inoculate M. oryzae for observing the infection process, however, this method is a time-consuming and high technical work. Here, we describe an easier solution to observe M. oryzae infection process on barley leaves.
Keywords: Magnaporthe oryzae Rice blast fungus Barley leaf epidermis Inoculation Infection process Plant-pathogen interactions
Background
The filamentous fungus Magnaporthe oryzae can cause destructive rice blast and wheat blast diseases, which can also infect barley (Kohli et al., 2011; Dean et al., 2012). M. oryzae has been studied as a model to understand fungal-plant interactions (Yan and Talbot et al., 2016). This fungus initiates its infection by attaching the conidium to host surface, then the conidium germinates and forms a dome-shaped appressorium, by which it can penetrate into host cell for colonization (Wilson and Talbot, 2009). In host cells, the fungus colonizes as a biotrophic manner by forming bulbous and branched infection hyphae to interact with host defense system (Kankanala et al., 2007). M. oryzae sequentially invade living host cells and finally transformed into necrotrophic growth, during which the lesions appear and sporulation occurs. In order to study the fungal infection mechanism, or protein functions during infection, or plant defense reaction, it is required to observe cellular infection process of different strains in host cells. A rice leaf sheath method has been commonly used to observe the infection process (Koga et al., 2004), however, this method needs to waste a long time to prepare the rice leaf sheath, and the inoculation and sample preparation need a great deal of experience. Because barley is also the host of M. oryzae, and its leaf epidermis is easy for tearing, so we found that barley leaf epidermis method is an effective and simple method to observe the infection process of M. oryzae.
Materials and Reagents
Petri dishes (6 cm and 9 cm diameters, ASONE)
Lens paper (Fisher Scientific)
Filter paper (Whatman)
Medical gauze (Angyang Medical)
Absorbent paper (Kimberly-Clark)
1.5 ml tubes (Eppendorf)
Tips (10 μl, 200 μl and 1,000 μl, Axygen)
Blades (Dexter Russell Cutlery, catalog number: 73-C )
Glass slides (Fisher Scientific, catalog number: 12-550-343 )
Coverslips (Fisher Scientific, catalog number: 12-547 )
Pots (10 cm in diameter and 15 cm in height)
Cultivated land soil
Magnaporthe oryzae strains
Note: The M. oryzae strains are maintained on dried filter paper pieces stock in -80 °C for long-term storage.
Barley seeds (Hordeum vulgare, cv E9)
Sterilized water (Milli-Q)
Boiled oatmeal filtrate
Tomato juice
Agar (Sigma-Aldrich, catalog number: 17209 )
Tween 20 (Sigma-Aldrich, catalog number: P9416 )
Oatmeal tomato agar (OTA) solid media (see Recipes)
0.025% Tween 20 (see Recipes)
Equipment
Scissor, tweezers, spreader
Pipettes (Eppendorf, catalog numbers: 3124000016 [0.5-10 µl], 3124000032 [20-200 µl], and 3124000075 [100-1,000 µl])
Incubation chamber (28 °C) for fungal growth and barley germination (Biolab Scientific, model: BIFG-101 )
Hemocytometer (Marienfeld, catalog number: 0650030 )
Greenhouse capable of temperature and humidity control for growing barley
Optical microscope (Olympus, model: CX23 )
Fluorescence microscope (Leica Microsystems, model: Leica DM2500 )
Juice extractor
Autoclave
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, X. (2018). Infection Process Observation of Magnaporthe oryzae on Barley Leaves. Bio-protocol 8(9): e2833. DOI: 10.21769/BioProtoc.2833.
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Category
Microbiology > Microbe-host interactions > Fungus
Plant Science > Plant immunity > Host-microbe interactions
Cell Biology > Cell-based analysis > Fungal infection
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2,834 | https://bio-protocol.org/exchange/protocoldetail?id=2834&type=0 | # Bio-Protocol Content
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Evaluation of the Condition of Respiration and Photosynthesis by Measuring Chlorophyll Fluorescence in Cyanobacteria
TO Takako Ogawa
K Kintake Sonoike
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2834 Views: 7017
Reviewed by: Alizée MalnoëShai SAroussi
Original Research Article:
The authors used this protocol in Mar 2015
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The authors used this protocol in:
Mar 2015
Abstract
Chlorophyll fluorescence measurements have been widely used to monitor the condition of photosynthesis. Furthermore, chlorophyll fluorescence from cyanobacteria reflects the condition of respiration, since cyanobacterial photosynthesis shares several components of electron transport chain with respiration. This protocol presents the method to monitor the condition of both photosynthesis and respiration in cyanobacteria simply by measuring chlorophyll fluorescence in the dark and in the light with pulse amplitude modulation (PAM) chlorophyll fluorometer.
Keywords: Cyanobacteria Chlorophyll fluorescence Non-photochemical quenching Photosynthesis Respiration
Background
Chlorophyll fluorescence measurements have been widely used to monitor the condition of photosynthesis in many photosynthetic organisms (Krause and Weis, 1991; Govindjee, 1995). In the case of cyanobacteria, the photosynthetic prokaryotes, chlorophyll fluorescence can be affected not only by the condition of photosynthesis but also by that of other metabolic pathways due to possible interactions among metabolic pathways within the cells. In particular, photosynthesis and respiration share several components of electron transport chain, such as plastoquinone (PQ) in cyanobacteria (Aoki and Katoh, 1982; Peschek and Schmetterer, 1982). The redox state of the PQ pool influences the yield of chlorophyll fluorescence through the regulation of state transition (Mullineaux and Allen, 1986; Mullineaux et al., 1997), which is the main component of the non-photochemical quenching in cyanobacteria (Campbell and Öquist, 1996). Thus, the cyanobacterial respiratory chain directly affects chlorophyll fluorescence especially in the dark, where photosynthesis is not active.
Due to the influence of respiration, chlorophyll fluorescence should be measured with caution in order to estimate photosynthesis precisely in cyanobacteria (Ogawa et al., 2013). On the other hand, the involvement of both photosynthesis and respiration in cyanobacterial chlorophyll fluorescence allows the estimation of not only photosynthesis but also respiration. In this protocol, we provide the method to monitor the condition of respiration and photosynthesis in cyanobacteria through the analysis of NPQ, the chlorophyll fluorescence parameter reflecting the level of non-photochemical quenching, measured in the dark (NPQDark) and under low light (NPQLL).
Materials and Reagents
Cyanobacteria
Note: The cyanobacterium Synechocystis sp. PCC 6803 is grown at 30 °C in BG11 medium (Rippka et al., 1979), buffered with 20 mM TES-KOH (pH 8.0) or 20 mM CHES-KOH (pH 9.0) and bubbled with air for 24 h under continuous illumination using fluorescent lamps.
Ferric ammonium citrate (Wako Pure Chemical Industries, catalog number: 092-00802 )
Na2EDTA·2H2O (Wako Pure Chemical Industries, catalog number: 345-01865 )
Sodium nitrate (NaNO3) (Wako Pure Chemical Industries, catalog number: 192-02555 )
Dipotassium hydrogenphosphate (K2HPO4) (Wako Pure Chemical Industries, catalog number: 164-04295 )
Magnesium sulfate (MgSO4) (anhydrous) (Wako Pure Chemical Industries, catalog number: 132-00435 )
Calcium chloride (CaCl2) (Wako Pure Chemical Industries, catalog number: 038-24985 )
Sodium carbonate (Na2CO3) (Wako Pure Chemical Industries, catalog number: 199-01585 )
Boric acid (H3BO4) (Wako Pure Chemical Industries, catalog number: 029-02191 )
Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Wako Pure Chemical Industries, catalog number: 139-00722 )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Wako Pure Chemical Industries, catalog number: 264-00402 )
Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Wako Pure Chemical Industries, catalog number: 039-04412 )
Disodium molybdate(VI) dihydrate (Na2MoO4·2H2O) (Wako Pure Chemical Industries, catalog number: 196-02472 )
Sulfuric acid (Wako Pure Chemical Industries, catalog number: 192-04696 )
Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Wako Pure Chemical Industries, catalog number: 031-03752 )
TES (Wako Pure Chemical Industries,, catalog number: 340-02655 )
Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (TCI, catalog number: D1328 )
Note: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) is an inhibitor of electron transport from QA to QB in photosystem II (PSII), thus oxidizing PQ pool under illumination. DCMU is used to determine maximum chlorophyll fluorescence level (Fm). We dissolve DCMU in ethanol (see below), and the stock solution at the concentration of 10 mM is added to the sample (equivalent to the final concentration of 20 μM) to determine the Fm level. DCMU solution can be stored for months in a freezer at -20 °C.
Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
BG11 stock solutions (see Recipes)
BG11 medium (see Recipes)
10 mM DCMU solution in ethanol (see Recipes)
Equipment
Test tubes (IWAKI, catalog number: TEST30NP )
Spherical micro quantum sensor (Heinz Walz, model: US-SQS/L )
Note: The light meter and the spherical micro-quantum sensor are used for measuring photon flux density of growth light and actinic light of WATER-PAM. It is advisable to use spherical micro-sensor for monitoring the photon flux density of actinic light of WATER-PAM, since the light illuminate samples from multiple directions.
Light meter (LI-COR, model: LI-250A )
Fluorometer (Heinz Walz, model: WATER-PAM )
Note: WATER-PAM (Heinz Walz, http://www.walz.com/products/chl_p700/water-pam/introduction.html) is a pulse amplitude modulation (PAM) fluorometer designed for measuring aquatic samples with low chlorophyll content. We use the Red LED type of the fluorometer with the emitter-detector unit of CUVETTE Version. The Red LED type of WATER-PAM is equipped with 3 LEDs peaking at 650 nm for the measuring light, 12 LEDs peaking at 660 nm for the actinic light as well as for the saturating pulse, and 3 LEDs peaking at 460 nm for blue light, which preferentially excite photosystem I (PSI) in cyanobacteria.
Quartz cuvette for WATER-PAM (Heinz Walz, model: WATER-K )
Spectrophotometer (JASCO, model: V-650 )
Note: The optical density of cell cultures at 750 nm is determined by the spectrophotometer. Any other common spectrophotometer can be used for this purpose.
Software
PC software ‘WinControl’ (WALZ, ver.3.22)
Note: WATER-PAM is operated from the PC software‘WinControl’.
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ogawa, T. and Sonoike, K. (2018). Evaluation of the Condition of Respiration and Photosynthesis by Measuring Chlorophyll Fluorescence in Cyanobacteria. Bio-protocol 8(9): e2834. DOI: 10.21769/BioProtoc.2834.
Download Citation in RIS Format
Category
Plant Science > Plant physiology > Photosynthesis
Microbiology > Microbial physiology > Photosynthesis
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2,835 | https://bio-protocol.org/exchange/protocoldetail?id=2835&type=0 | # Bio-Protocol Content
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An Image-based Assay for High-throughput Analysis of Cell Proliferation and Cell Death of Adherent Cells
Paula Szalai
Nikolai Engedal
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2835 Views: 9367
Edited by: Alessandro Didonna
Reviewed by: Michela PeregoSilvia Caggia
Original Research Article:
The authors used this protocol in Dec 2017
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The authors used this protocol in:
Dec 2017
Abstract
In this protocol, we describe a method to monitor cell proliferation and death by live-cell imaging of propidium iodide (PI)-stained adherent mammalian cells. PI is widely used to assess cell death. However, it is usually used in end-point assays. Recently, we implemented the use of PI for real-time cell death assessment by automated imaging. Cells are seeded in a 96-well format, and after attachment, the treatments are added directly to the wells together with PI. Thereafter, cells are subjected to automated time-lapse imaging and quantification by computer software. Combined analyses of phase-contrast and fluorescence images allow assessment of treatment effects on cell proliferation as well as the extent and kinetics of cell death.
Keywords: Cell death Cell proliferation Live-cell imaging Propidium iodide Thapsigargin Time-lapse imaging
Background
A variety of cell-based assays are available to determine cell death, but most of them, including the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, crystal blue staining, and various flow cytometry-based methods, have the limitation of being end-point assays. Recently, we employed propidium iodide (PI) for live-cell assessment of cell death using an automated fluorescence imaging system (IncuCyte ZOOM from Essen Bioscience) (Sehgal et al., 2017). By combined analysis of phase-contrast images, cytostatic and cytotoxic effects can be simultaneously detected. This method proved to be reliable and reproducible, as well as very simple and cheap. In our recent publication, we used it in three different cancer cell lines (LNCaP, PC3 and MCF7) to efficiently determine and compare the toxicities of various drug analogs of the ER Ca2+ pump inhibitor thapsigargin (Tg) (Sehgal et al., 2017). The use of PI for real-time analysis of cell death has been described and validated by others (Wlodkowic et al., 2009; Zhao et al., 2010), but the analysis methods employed previously (e.g., flow cytometry, or lab-on-chip platforms) are more laborious and time-consuming than the simple analysis method we present here. In short, we quantify cell death using the integrated algorithms of the IncuCyte ZOOM software to calculate the confluence of PI-positive (red-fluorescent) cells and divide that by the total cell confluence (obtained by analysis of phase-contrast images). This simple analysis approach minimizes the time and efforts needed for data processing and analysis, and increases the throughput of the method. We have validated the method for cell death assessment through a side-by-side comparison with flow cytometry-based end-point measurements of PI-stained cells.
Materials and Reagents
In the current protocol we use:
96-well tissue culture plates (Corning, Falcon®, catalog number: 353072 )
Sterile 50 ml tubes (VWR, catalog number: 525-0402 )
0.2 µm filter with PES membranes (VWR, catalog number: 514-0073 )
50 ml syringe (VWR, catalog number: 613-2053 )
LNCaP cells (ATCC, catalog number: CRL-1740 )
PC3 cells (ATCC, catalog number: CRL-1435 )
RPMI1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21875 )
Fetal bovine serum (Sigma-Aldrich, catalog number: F7524 ; use at 10% final concentration in RPMI 1640 medium to make complete growth medium)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
0.25% Trypsin-EDTA (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 20012 )
Propidium Iodide (Merck, Calbiochem®, catalog number: 537059 )
Thapsigargin (Sigma-Aldrich, catalog number: T9033 )
Propidium iodide, 1 mg/ml stock solution in PBS (see Recipes)
Thapsigargin (Tg), 5 mM stock solution in DMSO (see Recipes)
Equipment
Multi-Channel pipette, 8-channel, 30-300 µl (Eppendorf, catalog number: 3122000051 )
Autoflow IR Direct Heat CO2 Incubator (NuAire, model: NU-5510E )
IncuCyte ZOOM live-cell analysis system (Essen Bioscience, model: IncuCyte® ZOOM )
FACSCanto II Flow Cytometer (BD, BD Bioscience, model: FACSCanto II )
Software
IncuCyte ZOOM software (Essen Bioscience)
Flow cytometry Software (Perttu Terho)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Szalai, P. and Engedal, N. (2018). An Image-based Assay for High-throughput Analysis of Cell Proliferation and Cell Death of Adherent Cells. Bio-protocol 8(9): e2835. DOI: 10.21769/BioProtoc.2835.
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Category
Cancer Biology > Cell death > Cell biology assays
Cell Biology > Cell imaging > Live-cell imaging
Cell Biology > Cell viability > Cell death
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2,836 | https://bio-protocol.org/exchange/protocoldetail?id=2836&type=0 | # Bio-Protocol Content
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The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines
Morten Luhr
FS Frank Sætre
Nikolai Engedal
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2836 Views: 9604
Edited by: Alessandro Didonna
Reviewed by: Pia GiovannelliManasi K. Mayekar
Original Research Article:
The authors used this protocol in Oct 2013
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Original research article
The authors used this protocol in:
Oct 2013
Abstract
Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw conclusions regarding autophagic cargo flux. Here, we provide a protocol to quantitatively assess bulk autophagic flux by employing the long-lived protein degradation assay. Our procedure, which traces the degradation of 14C valine-labelled proteins, is simple and quick, allows for processing of a relatively large number of samples in parallel, and can in principle be used with any adherent cell line. Most importantly, it enables quantitative measurements of endogenous cargo flux through the autophagic pathway. As such, it is one of the gold standards for studying autophagic activity.
Keywords: Long-lived protein degradation Autophagy Autophagic flux Endogenous cargo Quantitative assay Pulse-chase Valine 14C radioactivity
Background
Pulse-chase labelling approaches have been used to study protein turnover for decades. In the long-lived protein degradation (LLPD) assay described here, the proteome of cells in culture is radiolabelled with 14C valine and chased in order to follow the decline in radioactive proteins as readout of protein degradation. After an initial chase period, the cells are washed to eliminate the degradation products of short-lived proteins, which predominantly result from proteasomal activity. Thereafter, a second chase is initiated, and proper controls are included in order to monitor the autophagic degradation of long-lived proteins. We recently used this method to discover that the calcium-modulating compounds thapsigargin and A23187, which based on results with autophagic markers were previously widely believed to activate autophagy, actually completely block bulk autophagic flux (Engedal et al., 2013). The starting material used in that and previous protein degradation protocols was derived from cells grown in 6-well plates (Bauvy et al., 2009; Engedal et al., 2013) or more (Ronning et al., 1979; Seglen et al., 1979), or involved relatively high amounts of radioactivity (Mizushima et al., 2001; Fuertes et al., 2003a), which is expensive. Recently, we have downscaled and simplified the LLPD protocol to the validated time- and cost-efficient version that we present here.
An overview of the method is shown in Figure 1. To label proteins with radioactive valine, cells are seeded in 24-well plates in complete medium supplemented with 14C valine. As proteins are synthesised, they incorporate amino acids, including the 14C valine present in the medium, and therefore the amount of long-lived 14C valine-labelled proteins increases with time (Figure 1, first part of the curve). Valine is an optimal amino acid to use in the LLPD method, since it is a poorly metabolised amino acid, is well tolerated at high doses, and does not influence autophagy or protein degradation rates (Seglen et al., 1979). Moreover, free valine is readily exchanged over the plasma membrane (Seglen and Solheim, 1978), enabling efficient wash-out of released 14C valine. After 2-3 days of labelling, unincorporated 14C valine is removed by a simple wash procedure, and the cells receive new medium supplemented with a high concentration of non-radioactive (‘cold’) valine (‘chase medium’). The large surplus of cold valine prevents reincorporation of released 14C valine. Thus, from this point on the presence of free 14C valine is a result of endogenous protein degradation. After a chase period of 18 h, the free 14C valine that has been produced by degradation of short-lived proteins (predominantly due to proteasomal degradation) is washed out. Next, a second chase period, which we call the ‘sampling period’, is initiated along with experimental treatments and proper controls. Generally, we use a sampling period of 2-6 h to monitor the degradation of long-lived proteins. At the end of the sampling period, trichloroacetic acid (TCA) is added to precipitate intact proteins. The TCA-soluble fraction of degraded proteins (containing free amino acids and small peptides) is separated from the TCA-insoluble fraction (containing intact proteins) by centrifugation, and the radioactivity in each fraction is measured by liquid scintillation counting. This allows calculation of the rate of long-lived protein degradation in the sampling period, expressed as the percentage of radioactivity in the TCA-soluble fraction versus the total amount of radioactivity in the TCA-soluble and–insoluble fractions, divided by the duration of the sampling period (Figure 1).
Figure 1. Overview of the long-lived protein degradation (LLPD) assay. During the labelling period (2-3 days), the amount of radioactive long-lived proteins increases with time. Thereafter, an 18 h chase period allows for degradation of the short-lived proteins and subsequent elimination of the released 14C valine by a washing step. Consequently, only the degradation of long-lived proteins is followed in the 2-6 h sampling period. Compared to cells kept in complete, nutrient-rich medium (red line), incubating cells with EBSS starvation medium or the mTOR-inhibitor Torin1 produces a very strong degradation of long-lived proteins in the sampling period, due to enhanced bulk autophagy (green line). Autophagic-lysosomal LLPD can be revealed by treatment with the lysosomal inhibitor Bafilomycin A1 (Baf) or RNAi-mediated silencing of key ATGs (siATGs) (yellow line), whereas the contribution to LLPD from the proteasome can be assessed by treatment with proteasomal inhibitors like MG132 (purple line). Blocking both autophagic-lysosomal and proteasomal activity simultaneously will abrogate both main sources of LLPD, thus resulting in minimal loss of 14C-labelled intact protein (black line). Note that the rise and fall in the curves are schematic and purely intended for illustrative purposes–they are not intended to indicate exact details in the kinetics of long-lived protein labelling and/or degradation. See text for a more detailed description of each of the protocol steps and for representative examples of data.
Materials and Reagents
In the current protocol we use:
Pipette tips (Thermo Fisher ART Barrier tips) (VWR, catalog numbers: 732-2223 (0.5-20 µl), 732-2207 (1-200 µl), and 732-2355 (100-1,000 µl))
75 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353136 )
24-well tissue culture plates (Corning, Falcon®, catalog number: 353047 )
Microcentrifuge tubes (VWR, catalog number: 211-2130 )
Scintillation vials (PerkinElmer, catalog number: 6000292 )
Nitrile gloves (VWR, catalog number: 112-2372 )
50 ml tube (VWR, catalog number: 525-0402 )
0.45 µm filter (VWR, catalog number: 514-0075 )
LNCaP cells (ATCC, catalog number: CRL-1740 )
U2OS cells (ATCC, catalog number: HTB-96 )
VCaP cells (ATCC, catalog number: CRL-2876 )
Huh7 cells (Nakabayashi et al., 1982) (Kindly provided by Dr. Line M. Grønning-Wang, Oslo, Norway)
PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 20012019 )
0.25% Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
RPMI 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 21875091 )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
[1-14C] L-valine, 45 mCi/mmol, 0.1 mCi/ml (Vitrax, catalog number: VC 308 )
Poly-D-lysine (Sigma-Aldrich, catalog number: P6407-10X5MG )
For RNAi reverse transfection:
Lipofectamine RNAiMAX (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
Ambion SilencerTM select siRNAs (negative control ‘siCtrl’, Thermo Fisher Scientific, InvitrogenTM, catalog number: 4390843 ; siULK1, s15964; siULK2, s18706)
Opti-MEM reduced serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11058021 )
Earle’s balanced salt solution (EBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 24010043 )
Scintillation liquid (PerkinElmer, catalog number: 6013199 )
DMSO (Sigma-Aldrich, catalog number: D2650 )
Bafilomycin A1 (Enzo Life Sciences, catalog number: BML-CM110-0100 )
Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514 )
SAR-405 (Magento, ApexBio, catalog number: A8883 )
Torin1 (Tocris Bioscience, catalog number: 4247 )
Non-radioactive L-valine (Sigma-Aldrich, catalog number: V0513 )
Bovine serum albumin (BSA) (VWR, catalog number: 422361V )
Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T0699 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
200 mM cold L-valine (see Recipes)
RPMI 1640/10%FBS (see Recipes)
1 mg/ml poly-D-lysine (see Recipes)
1 mM Torin1 (see Recipes)
PBS/2%BSA (see Recipes)
25% TCA (see Recipes)
0.2 M KOH (see Recipes)
0.2 mM Bafilomycin A1 (see Recipes)
160 mM NH4Cl (see Recipes)
Equipment
Pipettes (FinnpipetteTM F2 GLP Kit) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4701070 )
Humidified incubator
Tube rotator (VWR, catalog number: 444-0502 )
Plate shaker (Grant Instruments, model: PMS-1000i )
Magnetic stirrer (IKA, catalog number: 0003810001 )
Autoflow IR Direct Heat CO2 incubator (NuAire, model: NU-5510E )
Vortexer (Denville Scientific, catalog number: S7030 )
Benchtop centrifuge with cooling (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75002430 )
Scintillation counter (Liquid Scintillation Analyzer) (Packard, model: 1600 TR )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Luhr, M., Sætre, F. and Engedal, N. (2018). The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines. Bio-protocol 8(9): e2836. DOI: 10.21769/BioProtoc.2836.
Download Citation in RIS Format
Category
Biochemistry > Protein > Degradation
Cell Biology > Cell-based analysis > Autophagic activity
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2,837 | https://bio-protocol.org/exchange/protocoldetail?id=2837&type=1 | # Bio-Protocol Content
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Peer-reviewed
Nuclear Transformation of Chlamydomonas reinhardtii by Electroporation
Tyler M Wittkopp
Published: May 5, 2018
DOI: 10.21769/BioProtoc.2837 Views: 9659
Edited by: Adam Idoine
Reviewed by: Ru Zhang
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Abstract
The unicellular green alga Chlamydomonas reinhardtii is an important model organism for studying photosynthesis, acclimation to abiotic stress, cilia biology, and many other biological processes. Many molecular biology tools exist for interrogating gene function including the ability to easily transform the nuclear genome of Chlamydomonas. While technical advances such as TALENs, ZFNs and CRISPR are making it easier to precisely edit the nuclear genome, the efficiency of such methods in Chlamydomonas is at present very low. In contrast, random insertion by nuclear transformation tends to be a much more efficient process. This protocol describes a method for transformation of the Chlamydomonas nuclear genome by electroporation. The protocol requires at least 3 days of work and generally results in the appearance of small colonies within 1-2 weeks.
Keywords: Algae Nuclear transformation Electroporation Fluorescent fusion protein Venus mCherry FLAG tag His tag Paromomycin Hygromycin
Background
Numerous molecular, genetic and genomic resources make Chlamydomonas reinhardtii (Chlamydomonas hereafter) an excellent model organism for studies on diverse biological processes. Many techniques have been developed to transform the Chlamydomonas nucleus, chloroplast and mitochondria including particle bombardment (Boynton et al., 1988), glass bead transformation (Kindle, 1990), and electroporation (Shimogawara et al., 1998). Nuclear mutants may be generated by exposure of Chlamydomonas cells to physical or chemical mutagens (e.g., UV light or ethyl methanesulfonate), but are often obtained by random insertional mutagenesis of transgenic DNA. Since the efficiency of homologous recombination for nuclear transformation in Chlamydomonas is very low (Zorin et al., 2009; Jinkerson and Jonikas, 2015), transformed DNA is generally integrated into the nuclear genome at random sites. A number of techniques exist for subsequently identifying the insertion sites of the ectopic DNA including classical genetic mapping (Rymarquis et al., 2005), TAIL-PCR (Dent et al., 2005), and next-generation sequencing of individual mutants (Dutcher et al., 2012) or large mutant libraries (Zhang et al., 2014; Li et al., 2016). While recent technical advances have led to improvements in targeted genome editing in Chlamydomonas using CRISPR/Cas9 (Baek et al., 2016; Shin et al., 2016; Ferenczi et al., 2017; Greiner et al., 2017) and zinc-finger nucleases (Sizova et al., 2013; Greiner et al., 2017), random insertional mutagenesis is still a preferred method to generate mutant libraries for forward and reverse genetics.
This protocol describes a detailed method for nuclear transformation of Chlamydomonas by electroporation. It can be used to generate random insertion mutants using a plasmid fragment conferring antibiotic resistance (Jinkerson and Jonikas, 2015) or for the expression of fluorescent fusion proteins using well-established, publically-available expression vectors. Once a suitable DNA fragment has been obtained or generated, the transformation protocol takes two days and generally results in visible, isolated colonies within 1-2 weeks.
Materials and Reagents
Pipette tips with filters
Sterile 0.6 ml microcentrifuge tubes
Sterile 15 ml centrifuge tubes
Sterile 50 ml centrifuge tubes
0.4 cm gap electroporation cuvettes (Bio-Rad Laboratories, catalog number: 1652088 )
Petri dishes
Blue Sharpie (permanent) marker pen
Sterile plastic inoculating loops (VWR, catalog number: 12000-810 )
Parafilm
Optional: plasmid pLM005 (available from the Chlamydomonas Resource Center, www.chlamy.org)
Optional: plasmid pLM006 (available from the Chlamydomonas Resource Center, www.chlamy.org)
Optional: plasmid pMO449 (available from the Chlamydomonas Resource Center, www.chlamy.org)
Optional: wild-type strain CC-1690 21gr+ (available from the Chlamydomonas Resource Center, www.chlamy.org)
70% ethanol
Canned air (Fisher Scientific, catalog number: 23-022-523 )
Tris base (Biopioneer, catalog number: C0060 )
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3306 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
Ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA·Na2·2H2O) (Sigma-Aldrich, catalog number: E5134 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
Ammonium molybdate tetrahydrate [(NH4)6Mo7O24·7H2O] (Sigma-Aldrich, catalog number: A7302 )
Sodium selenite (Na2SeO3) (Sigma-Aldrich, catalog number: S5261 )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z4750 )
Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: M3634 )
Iron(III) chloride hexahydrate (FeCl3·6H2O) (Sigma-Aldrich, catalog number: F2877 )
Copper(II) chloride dihydrate (CuCl2·2H2O) (Sigma-Aldrich, catalog number: C3279 )
Glacial acetic acid (Fisher Scientific, catalog number: A38-212 )
Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
Agar (Caisson Laboratories, catalog number: A038 )
Paromomycin sulfate salt (Paro) (Sigma-Aldrich, catalog number: P8692 )
Hygromycin B (Hyg) 50 mg/ml solution (Clontech, catalog number: 631309 )
Stock solutions (see Recipes)
1 M Tris Base (1 L, 50x)
Solution A for TAP (500 ml, 100x)
125 mM EDTA·Na2 pH 8.0 (300 ml)
285 µM (NH4)6Mo7O24 (250 ml)
1 mM Na2SeO3 (250 ml)
100 mg/ml paromomycin (1 ml)
Micronutrient stock solutions (see Recipes)
25 mM EDTA·Na2 (250 ml)
28.5 µM (NH4)6Mo7O24 (250 ml)
0.1 mM Na2SeO3 (250 ml)
Zn-EDTA (250 ml)
Mn-EDTA (250 ml)
Fe-EDTA (250 ml)
Cu-EDTA (250 ml)
TAP liquid medium (see Recipes)
TAP 40 mM sucrose (see Recipes)
TAP + 20 µg/ml Paro selective agar medium (see Recipes)
TAP + 25 µg/ml Hyg selective agar medium (see Recipes)
*Note: Materials used for generating plasmids of interest (polymerases, restriction enzymes, ligases, buffers, etc.) are not described here.
Equipment
Sterile 250 ml culture flasks (e.g., Corning, catalog number: 70980-250 )
Sterile 1 or 2 L culture flasks (e.g., DWK Life Sciences, Kimble®, catalog numbers: 26500-1000 or 26500-2000 )
Pipettes
Centrifuge (e.g., Eppendorf, model: 5810 R ) and microcentrifuge (e.g., Eppendorf, model: 5424 )
NanoDrop 2000 (or similar) spectrophotometer
Hemacytometer (e.g., Sigma-Aldrich, catalog number: Z359629 ) or automated cell counter (e.g., Bio-Rad Laboratories, model: TC20 )
Water bath with thermometer
Biosafety cabinet (with optional UV light)
Note: A laminar flow hood can also be used
Gene Pulser XCell Electroporator (Bio-Rad Laboratories, catalog number: 1652660 )
Tube shaker (e.g., Thermo Fisher Scientific, catalog number: T415110Q )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wittkopp, T. M. (2018). Nuclear Transformation of Chlamydomonas reinhardtii by Electroporation. Bio-101: e2837. DOI: 10.21769/BioProtoc.2837.
Download Citation in RIS Format
Category
Plant Science > Phycology > DNA > Transformation
Plant Science > Phycology > Nuclear transformation
Molecular Biology > DNA > Transformation
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2,838 | https://bio-protocol.org/exchange/protocoldetail?id=2838&type=1 | # Bio-Protocol Content
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Peer-reviewed
Isolation of Genomic DNA from Chlamydomonas reinhardtii
Tyler M Wittkopp
Published: May 5, 2018
DOI: 10.21769/BioProtoc.2838 Views: 13419
Edited by: Adam Idoine
Reviewed by: Yunbing Ma
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Abstract
Chlamydomonas reinhardtii is a soil-dwelling eukaryotic green alga that is widely used as a laboratory model organism for research on photosynthesis, ciliary biology, lipid metabolism and many other aspects of cell biology and physiology. With sequenced nuclear, chloroplast and mitochondrial genomes, Chlamydomonas is also an excellent organism for genetics and genomics research. This protocol describes the isolation of genomic DNA from Chlamydomonas using a standard phenol:chloroform extraction method followed by ethanol precipitation. The protocol requires minimal lab materials, takes approximately 4 h to complete, and can also be used for isolation of genomic DNA from other eukaryotic green algae.
Keywords: Algae Genomic DNA Phenol/chloroform extraction Nucleic acid DNA purification
Background
Isolating nucleic acid is a critical first step for cloning and sequencing genetic material and provides the basis for diverse molecular biological studies ranging from gene expression to gene evolution. A number of protocols exist for isolating DNA from algae (Weeks et al., 1986; Fawley and Fawley, 2004; HwangBo et al., 2010). Generally, cells are pelleted by centrifugation and lysed in buffer containing detergents such as SDS to solubilize membranes. This is followed by at least one extraction in phenol:chloroform and at least one extraction in chloroform. In some cases, an RNase treatment step is performed to degrade RNA. DNA is then precipitated by addition of ethanol or isopropanol and incubating on ice or in the freezer. After pelleting and washing the precipitated DNA, it is usually resuspended in water or buffer (e.g., Tris-EDTA) and quantified spectrophotometrically at 260 nm.
The protocol described herein makes use of two phenol/chloroform/isoamyl alcohol extractions and two chloroform/isoamyl extractions, with an RNase treatment step in between. The addition of isoamyl alcohol to the organic solvents prevents foaming and stabilizes the interphase, which contains a high concentration of coagulated proteins. Importantly, this protocol results in high quality genomic DNA that is suitable for downstream applications such as cloning and sequencing.
Materials and Reagents
Pipette tips
1.5 ml microcentrifuge tubes
50 ml centrifuge tubes
Kimwipe
RNase A (50 mg/ml) (Sigma-Aldrich, catalog number: R6513 )
Phenol/chloroform/isoamyl alcohol pH 8.0 (25:24:1) (Sigma-Aldrich, catalog number: P2069 )
Notes:
Separates into two phases–use the bottom organic phase.
Use caution when handling this solution; work in a chemical hood and wear gloves, eye protection.
Chloroform/isoamyl alcohol (24:1) (Sigma-Aldrich, catalog number: 25666 )
Note: Use caution when handling this solution; work in a chemical hood and wear gloves, eye protection.
Absolute ethanol (Sigma-Aldrich, catalog number: 792780 )
70% ethanol
Nuclease-free water
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
Ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA·Na2·2H2O) (Sigma-Aldrich, catalog number: E5134 )
Ammonium molybdate tetrahydrate [(NH4)6Mo7O24·7H2O] (Sigma-Aldrich, catalog number: A7302 )
Sodium selenite (Na2SeO3) (Sigma-Aldrich, catalog number: S5261 )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z4750 )
Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: M3634 )
Iron(III) chloride hexahydrate (FeCl3·6H2O) (Sigma-Aldrich, catalog number: F2877 )
Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
Copper(II) chloride dihydrate (CuCl2·2H2O) (Sigma-Aldrich, catalog number: C3279 )
Tris base (Biopioneer, catalog number: C0060 )
Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3306 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
Glacial acetic acid (Fisher Scientific, catalog number: A38-212 )
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
DNA Extraction buffer (see Recipes)
Micronutrient stock solutions (see Recipes)
Stock solutions (see Recipes)
Tris-Acetate-Phosphate (TAP) growth medium (see Recipes)
Equipment
250 ml culture flasks (e.g., Corning, PYREX®, catalog number: 70980-250 )
Pipettes
Hemacytometer (e.g., Sigma-Aldrich, catalog number: Z359629 ) or automated cell counter (e.g., Bio-Rad Laboratories, model: TC20TM )
Basic light microscope (e.g., Amscope, catalog number: B100B-MS )
Vortex mixer (e.g., Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
Centrifuge (e.g., Eppendorf, model: 5810 R ) and microcentrifuge (e.g., Eppendorf, model: 5424 )
Freezer
Nano-Drop 1000/2000 spectrophotometer or Qubit 2.0 fluorometer
Optional: PCR thermal cycler
Autoclave
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wittkopp, T. M. (2018). Isolation of Genomic DNA from Chlamydomonas reinhardtii. Bio-101: e2838. DOI: 10.21769/BioProtoc.2838.
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Category
Plant Science > Phycology > DNA > Extraction
Plant Science > Plant molecular biology > DNA > DNA extraction
Molecular Biology > DNA > DNA extraction
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2,839 | https://bio-protocol.org/exchange/protocoldetail?id=2839&type=0 | # Bio-Protocol Content
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Peer-reviewed
Functional Evaluation of the Signal Peptides of Secreted Proteins
Weixiao Yin
YW Yufu Wang
TC Tao Chen
YL Yang Lin
CL Chaoxi Luo
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2839 Views: 10945
Reviewed by: Xiaolin Chen
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
Here, we describe a method that can be used to evaluate the function of predicted signal peptides. This method utilizes the yeast Saccharomyces cerevisiae YTK12 strain and pSUC2 vector in which the pSUC2 vector with fused predicted signal peptide is transformed into yeast. The function of the signal peptides can be evaluated by using different selective media and color reaction. In this protocol, we provide the detailed description of manipulation in order to implement easily.
Keywords: Secreted protein Signal peptide Function Yeast pSUC2 vector
Background
Microbial eukaryotes, such as fungi and oomycetes, secret abundant of proteins to play a variety of functions. Currently, the most widely used method for prediction of signal peptide from amino acid sequences is to use software SignalP (Petersen et al., 2011). The yeast YTK12 strain is invertase negative and pSUC2 vector contains invertase gene but lack Methionine (Met) and signal peptide sequence, thus YTK12 strain and pSUC2 vector were widely used for biological evaluation of peptide secretion (Jacobs et al., 1997; Oh et al., 2009). In addition, the color reaction could be used to verify the result, because the invertase enzymatic activity can be detected by the reduction of 2,3,5-Triphenyltetrazolium Chloride (TTC) to insoluble red colored 1,3,5-Triphenylformazan (TPF). Although the method has been generally used to evaluate the function of signal peptides, there is no specific and detailed information about this method (Oh et al., 2009; Song et al., 2015). Here, we describe a modified high-efficiency yeast transformation method (Gietz and Schiestl, 2007) and detailed protocol to evaluate the function of signal peptides, which will be the primary part for the secretory proteins research.
Materials and Reagents
Pipette tips
1.5 ml tubes
2 ml tubes
10 ml test tube
Millipore filter units, 0.22 µm (Millex-GP, Merck, catalog number: SLGP033RB )
Yeast YTK12 strain
pSUC2 vector
EcoRI restriction enzymes (Takara Bio, catalog number: 1040S )
XhoI restriction enzymes (Takara Bio, catalog number: 1094S )
Yeast extract (OXOID, catalog number: LP0021 )
Peptone (BD, DifcoTM, catalog number: 211677 )
Glucose (Sinopharm Chemical Reagent, catalog number: 10010518 )
Agar A (Sangon Biotech, catalog number: A600010 )
Adenine hemisulfate salt (Sigma-Aldrich, Vetec, catalog number: V900375 )
Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
EDTA (Sinopharm Chemical Reagent, catalog number: 10009617 )
NaOH (Sinopharm Chemical Reagent, catalog number: 10019718 )
Single-stranded carrier DNA (salmon sperm DNA, Solarbio, catalog number: D8030 )
LiAc (Sinopharm Chemical Reagent, catalog number: 30109760 )
PEG (Polyethylene glycol, Sigma-Aldrich, catalog number: P3640 )
DMSO (Dimethyl Sulfoxide, Sigma-Aldrich, catalog number: D5879 )
YNB (yeast nitrogen base without amino acids, BD, DifcoTM, catalog number: 291920 )
-Trp DO supplement (Clontech, catalog number: 630413 )
Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
Antimycin A (Sigma-Aldrich, catalog number: A8674 )
Raffinose (D-(+)-Raffinose pentahydrate, Bomei, CAS Number: 17629-30-0)
Sodium acetate (Sinopharm Chemical Reagent, catalog number: 10018718 )
Acetic acid (Sinopharm Chemical Reagent, catalog number: 10000218 )
KH2PO4 (Sinopharm Chemical Reagent, catalog number: 10017618 )
Na2HPO4 (Sinopharm Chemical Reagent, catalog number: 10020318 )
TTC (Tokyo Chemical Industry (TCI), CAS Number: 298-96-4)
YPD medium (1 L) (see Recipes)
1x TE Buffer (100 ml) (see Recipes)
Single-stranded carrier DNA (2 mg/ml) (see Recipes)
1.0 M LiAc (100 ml) (see Recipes)
50% (w/v) PEG (100 ml) (see Recipes)
CMD-W medium (1 L) (see Recipes)
Antimycin A stock solution (50 mg/ml) (see Recipes)
YPRAA medium (1 L) (see Recipes)
10 mM acetic acid-sodium acetate buffer (100 ml, pH = 4.7) (see Recipes)
Phosphate Buffer (150 ml) (see Recipes)
TTC Stock solution (2%) (see Recipes)
Equipment
Pipettes (Mettler-Toledo International, RAININ, model: XLS )
Incubator (ZHICHENG, model: ZXDP-B2050 )
Clean bench (AIRTECH, model: SW-CJ-2FD , catalog number: A11062689)
Water bath (Shanghai Jinghong Laboratory Instrument, model: DK-8D )
Vortex mixer (Kylin-Bell Lab Instruments, model: VORTEX-5 )
Incubator shaker (Changzhou Zhiborui Instrument Manufacturing, model: THZ-D )
Centrifuge (Eppendorf, model: 5424 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Yin, W., Wang, Y., Chen, T., Lin, Y. and Luo, C. (2018). Functional Evaluation of the Signal Peptides of Secreted Proteins. Bio-protocol 8(9): e2839. DOI: 10.21769/BioProtoc.2839.
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Category
Plant Science > Plant immunity > Host-microbe interactions
Molecular Biology > Protein > Detection
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Why The YTK12 strain and YTK12 carrying the empty pSUC2 vector can grow on YPRAA medium?
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284 | https://bio-protocol.org/exchange/protocoldetail?id=284&type=0 | # Bio-Protocol Content
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Glucose Production Assay in Primary Mouse Hepatocytes
Michihiro Matsumoto
MS Mashito Sakai
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.284 Views: 25872
Original Research Article:
The authors used this protocol in Mar 2012
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Abstract
Hepatic glucose production is a primary determinant of fasting hyperglycemia in type 2 diabetic patients. Glucagon-cAMP-PKA pathway increases, but insulin-PI3 kinase-Akt pathway suppresses glucose production. This assay aims to evaluate the ability of isolated mouse hepatocytes to release newly synthesized glucose mainly from lactate and pyruvate as the substrates (i.e. gluconeogenesis) under basal, cAMP-, or cAMP plus insulin-treated condition.
Materials and Reagents
Primary mouse hepatocytes
Medium199 (Life Technologies, Invitrogen™, catalog number: 11150-059 )
Fetal bovine serum (FBS)
bicinchoninic acid (BCA)
Penicillin-Streptomycin, Liquid (Life Technologies, Invitrogen™, catalog number: 15140-122 )
PBS+/+ (Sigma-Aldrich, catalog number: D8662 )
Dulbecco’s modified eagle’s medium (DMEM), without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate, powder (Sigma-Aldrich, catalog number: D5030 )
Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
Sodium L-lactate (Sigma-Aldrich, catalog number: L7022 )
Sodium pyruvate (Life Technologies, Invitrogen™, catalog number: 11360-070 )
100 mM MEM sodium pyruvate solution (100x), liquid (Life Technologies, Invitrogen™, catalog number: 11360-070 )
200 mM L-Glutamine (100x), liquid (Life Technologies, Invitrogen™, catalog number: 25030-081 )
1 M HEPES buffer solution (Life Technologies, Invitrogen™, catalog number: 15630-080 )
pCPT-cAMP (Sigma-Aldrich, catalog number: C3912 )
Insulin (Sigma-Aldrich, catalog number: I9278 )
Autokit glucose (Wako, catalog number: 439-90901 )
BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23227 )
HEPES
Glucose production buffer (see Recipes)
Equipment
iMark Microplate Absorbance Reader (Bio-Rad, catalog number: 168-1135 )
BD BioCoat™Collagen I 6-well Plates (BD Biosciences, catalog number: 356400 )
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Matsumoto, M. and Sakai, M. (2012). Glucose Production Assay in Primary Mouse Hepatocytes. Bio-protocol 2(21): e284. DOI: 10.21769/BioProtoc.284.
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Category
Biochemistry > Carbohydrate > Glucose
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2,840 | https://bio-protocol.org/exchange/protocoldetail?id=2840&type=0 | # Bio-Protocol Content
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Peer-reviewed
Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta
LZ Lijia Zheng
CZ Chao Zhang
Jianguo Wu
YL Yi Li
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2840 Views: 5504
Edited by: Arsalan Daudi
Reviewed by: Aswad Khadilkar
Original Research Article:
The authors used this protocol in Oct 2017
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Oct 2017
Abstract
MicroRNAs (miRNAs) play important roles in plant growth, development, and response to infection by microbes. Double-stranded RNA binding protein 1 (DRB1) facilitates the processing of primary miRNA transcripts into mature miRNAs. Recently, we found that NS3 protein encoded by rice stripe virus (RSV) associates with DRB1 and promotes miRNA biogenesis during RSV infection (Zheng et al., 2017). RNA co-immunoprecipitation (RIP) method was applied to identity association patterns among DRB1, NS3, and miRNA transcript.
Keywords: Rice stripe virus NS3 Double-stranded RNA binding protein Primary-miRNA miRNA Plant-microbe interaction
Background
MicroRNAs (miRNAs) are processed from their primary transcripts (pri-miRNAs) by the RNase III enzyme DICER-LIKE 1 (DCL1) with the help of the double-stranded RNA (dsRNA) binding protein HYPONASTIC LEAVES1 (DRB1/HYL1) and the zinc finger protein SERRATE (SE). Rice stripe virus (RSV) infection broadly perturbs miRNA accumulation. We found that RSV-encoding nonstructural protein 3 (NS3) promotes miRNA accumulation by downregulating pri-miRNAs through interaction with DRB1 in rice (Zheng et al., 2017). To reveal how NS3 enhances pri-miRNA processing, we used co-immunoprecipitation (Co-IP) to illustrate the relationship of NS3, DRB1 and pri-miRNA in vivo. This protocol contributes to understand association patterns between two proteins and one RNA transcript.
Materials and Reagents
Pipette (RNase free 1 ml, 0.2 ml and 0.02 ml, Axygen)
Miracloth (Merck, Calbiochem, catalog number: 475855 )
Centrifuge tube (1.5 ml, 2 ml and 50 ml) (Corning)
4- to 6- weeks old Nicotiana benthamiana (leaves, grow in green house)
Agrobacterium tumefaciens (EHA105 strain, preserved in our lab)
Expression plasmids: pEarleyGate202-DRB1, -mutant DRB1, pEarleyGate203-NS3, -mutant NS3 and pCAMBIA-artificial primary miRNA transcript, -mutant primary miRNA transcript (Constructed by ourselves)
Double-distilled or MilliQ water (ddH2O)
Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 70011069 )
Glycine (Sigma-Aldrich, catalog number: V900144 )
Liquid nitrogen
Anti-Myc (9E10) monoclonal antibodies (Sigma-Aldrich, catalog numbers: M4439 ) and mouse IgG control (Thermo Fisher Scientific, Invitrogen, catalog number: 02-6502 )
Protein G-agarose (Roche Diagnostics, catalog number: 11243233001 )
Trizol
Chloroform (Sigma-Aldrich, catalog number: 613304 )
GlycoBlueTM coprecipitant (15 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9516 )
Ethanol (Sigma-Aldrich, catalog number: E7023 )
Ethanol (Aladdin, catalog number: E111963 )
DNase I (Promega, catalog number: M6101 )
Sucrose (Sigma-Aldrich, catalog number: V900116 )
Ficoll 400 (Sigma-Aldrich, catalog number: F9378 )
Dextran T40 (Sigma-Aldrich, catalog number: 1179708 )
HEPES (Sigma-Aldrich, catalog number: RDD002 )
KOH
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 05892953001 )
DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: 43817 )
Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
NP-40 (Sigma Aldrich, catalog number: NP40S )
RNase inhibitor (RNaseOUT) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777019 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: V900312 )
Diethyl pyrocarbonate (Sigma-Aldrich, catalog number: 40718 )
Honda buffer (see Recipes)
High salt nuclear lysis buffer (see Recipes)
Dilution buffer (see Recipes)
IP buffer (see Recipes)
Equipment
Eppendorf pipettes suite (1 ml, 0.2 ml, 0.02 ml, 0.01 ml and 0.0025 ml)
Thermomixer C (Eppendorf, model: Thermomixer® C , catalog number: 5382000023)
Vacuum pump (FJC, model: 6912 )
Rotator (Glas-Col, model: 099A MR1512 )
Centrifuge (Eppendorf, models: 5424 R and 5804 R )
Pico Ultrasonicator (Diagenode, model: Bioruptor® Pico, model: 4486126 )
Vortex (Kylin-Bell Lab Instruments, model: VORTEX-5 )
Pestle
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zheng, L., Zhang, C., Wu, J. and Li, Y. (2018). Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta. Bio-protocol 8(9): e2840. DOI: 10.21769/BioProtoc.2840.
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Category
Microbiology > Microbe-host interactions > Virus
Plant Science > Plant molecular biology > Protein
Molecular Biology > RNA > RNA-protein interaction
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2,841 | https://bio-protocol.org/exchange/protocoldetail?id=2841&type=0 | # Bio-Protocol Content
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Nab Escaping AAV Mutants Isolated from Mouse Muscles
ZC Zheng Chai
RS R. Jude Samulski
CL Chengwen Li
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2841 Views: 6267
Edited by: Longping Victor Tse
Reviewed by: George William CarnellVikas Duhan
Original Research Article:
The authors used this protocol in Feb 2016
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Feb 2016
Abstract
Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These AAV chimeric capsid mutants enhanced Nab evasion from patient serum with a high muscle transduction efficacy. In this protocol, we describe the procedures for selection of the Nab-escaped AAV chimeric capsid, including isolation and characterization of Nab-escaping AAV mutants in mice muscle.
Keywords: AAV Nab-escaping AAV Chimeric capsid Mouse muscle
Background
Adeno-associated virus (AAV) vectors have been used in many preclinical studies and clinical trials. Many diseases have received eventual treatment using AAV gene therapy. However, the presence of neutralizing antibodies in circulation poses a major challenge for AAV vector application in future clinical trials. Many approaches have been explored to evade activities of Nab. Herein, we described the approach with directed evolution for selection of Nab-escaping mutants from an AAV shuffling library.
DNA shuffling is a powerful strategy for generating diverse mutants. Through successive rounds of phenotypic selection, DNA shuffling libraries were characterized by higher quality and more targeted diversification. High-throughput selection of capsid mutants from AAV shuffling libraries has been used as a promising strategy to explore AAV mutants with the abilities to target specific tissues and evade Nabs. However, most of these selecting methods were only tested in vitro; some studies even used rabbit anti-AAV2 sera or human intravenous immunoglobulin. The approach of in vivo selection of capsid mutants could provide a platform to generate more effective AAV mutants that not only escape Nab from patient serum but also enhance transduction in specific tissues.
Materials and Reagents
GeneMate individual 0.2 ml PCR tubes (BioExpress, catalog number: T-3035-1 )
BD Veo insulin syringes with BD Ultra-Fine 6 mm x 31G needle (BD, catalog number: 324910 )
CorningTM 96-Well Clear Bottom Black Polystyrene Microplates (Corning, catalog number: 3904 )
VWR® Tissue Culture 48-well Plate (VWR, catalog number: 10861-560 )
BALB/c mice, typically of 6 weeks old female mice
ElectroMAXTM DH10BTM Cells (Thermo Fisher Scientific, catalog number: 18290015 )
Adherent HEK 293 cells and Huh7 cells
MAX EfficiencyTM DH10BTM Cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18297010 )
JBS DNA-Shuffling Kit (Jena Bioscience, catalog number: PP-103 )
DNase I, RNase-free (1 U/µl) (Thermo Fisher Scientific, catalog number: EN0521 )
EDTA
QIAquick PCR Purification Kit (250) (QIAGEN, catalog number: 28106 )
Purified single-stranded AAV (any serotype) (Xiao et al., 1998)
PfuUltra High-Fidelity DNA polymerase (Agilent Technologies, Santa Clara, CA)
Wild type AAV2 plasmid psub201, plasmid pXR2 (RepCap plasmid) and pXX6-80 (a helper plasmid contains the genes E4, E2a and VA from adenovirus)
1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
Patient serum from clinical study for Duchenne Muscular Dystrophy (Bowles et al., 2012)
Adenovirus dl309
DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
SwaI (New England Biolabs, catalog number: R0604S )
XbaI (New England Biolabs, catalog number: R0145S )
Passive lysis buffer (Promega, catalog number: E1941 )
Luciferase Assay Substrate (Promega, catalog number: E151A )
XenoLight D-Luciferin - Bioluminescent Substrate (PerkinElmer, catalog number: 122799 )
25 mg/ml D-luciferin substrate (see Recipes)
Equipment
Pipettes
Bio-Rad Thermal Cyclers (Bio-Rad Laboratories, catalog number: 1709703 )
Tabletop centrifuge (Eppendorf, model: 5424 , catalog number: 022620401)
VICTOR Multilabel Plate Reader (PerkinElmer)
Small animal anesthesia system (Xenogen, XGI-8 Gas Anesthesia System)
IVIS Lumina In Vivo imaging system (PerkinElmer)
Software
Living Image software (PerkinElmer)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chai, Z., Samulski, R. J. and Li, C. (2018). Nab Escaping AAV Mutants Isolated from Mouse Muscles. Bio-protocol 8(9): e2841. DOI: 10.21769/BioProtoc.2841.
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Category
Immunology > Antibody analysis > Antibody function
Microbiology > Antimicrobial assay > Antiviral assay
Molecular Biology > Protein > Anti-microbial analysis
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2,842 | https://bio-protocol.org/exchange/protocoldetail?id=2842&type=0 | # Bio-Protocol Content
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Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
PM Prarthana Mohanraju
John van der Oost
MJ Martin Jinek
Daan C. Swarts
Published: Vol 8, Iss 9, May 5, 2018
DOI: 10.21769/BioProtoc.2842 Views: 19437
Edited by: Renate Weizbauer
Reviewed by: Rainer MelzerPeter E Burby
Original Research Article:
The authors used this protocol in Apr 2017
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Abstract
This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments.
Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli
Keywords: CRISPR-Cas Cas12a Cpf1 Protein purification
Background
Prokaryotic CRISPR-Cas immune systems provide protection against viruses and plasmids by using CRISPR RNAs (crRNAs) as a guide for sequence-specific targeting of foreign DNA or RNA (van der Oost et al., 2014; Marraffini, 2015). Class 1 CRISPR-Cas systems (comprising types I, III, and IV) typically form multi-subunit protein-crRNA effector complexes, while the class 2 systems (comprising types II, V, and VI) rely on single crRNA-guided effector nucleases for target interference (Mohanraju et al., 2016).
Effector nuclease enzymes from the Class 2 CRISPR-Cas systems have emerged as efficient and precise tools for genome editing and gene expression control (Mali et al., 2013; Doudna and Charpentier, 2014; Hsu et al., 2014). The widely used Cas9, which is the signature protein of type II systems, utilizes a dual guide RNA structure consisting of crRNA and a trans-activating crRNA (tracrRNA) for target recognition (Deltcheva et al., 2011). For genome editing purposes, the dual guide RNA is often replaced by a synthetic fusion of the mature crRNA and tracrRNA, resulting in a long single-molecule guide RNA (sgRNA) in which the individual RNAs are fused by a short linker sequence (Jinek et al., 2012). The sequence of the guide RNA allows binding of complementary DNA targets by base pairing with the target strand, while the other strand of the DNA is displaced. Upon finding a cognate DNA target, the HNH and RuvC nuclease domains of Cas9 mediate cleavage of the target and the displaced strand, respectively (Jinek et al., 2012; Karvelis et al., 2013).
More recently, another novel class 2 CRISPR-Cas nuclease with distinctive features has been identified in bacterial genomes: Cas12a (also known as Cpf1) (Makarova and Koonin, 2015; Zetsche et al., 2015; Shmakov et al., 2017). Cas12a utilizes a single crRNA guide for DNA targeting; it does not require a tracrRNA, resulting in a shorter gRNA sequence compared to the chimeric single-molecule guide RNAs (sgRNA) used by Cas9. While Cas9 requires RNase III-mediated processing of pre-crRNA or individual expression of sgRNAs for the formation of mature guide RNAs, Cas12a can process its own pre-crRNA. This pre-crRNA processing activity allows for simple multiplexing in Cas12a-mediated genome editing (Wang et al., 2017; Zetsche et al., 2017). Whereas Cas9 generates double stranded DNA breaks (DSBs) that are blunt ended, Cas12a generates staggered-end DSBs (Zetsche et al., 2015). Such overhangs can be utilized for overhang-based cloning (Li et al., 2016; Lei et al., 2017). Moreover, Cas9 typically recognizes a G-rich PAM sequence, while all Cas12a orthologues characterized to date recognize a T-rich PAM sequence (Zetsche et al., 2015). Taken together, these features make Cas12a a valuable addition to the genome editing toolbox.
Cas12a has been successfully repurposed for genome editing applications in mammalian cells (Zetsche et al., 2015; Kim et al., 2016a), mice (Hur et al., 2016; Kim et al., 2016b), rice (Endo et al., 2016; Hu et al., 2017; Xu et al., 2017), yeast (Verwaal et al., 2017; Swiat et al., 2017), zebrafish, xenopus (Moreno-Mateos et al., 2017), microalga (Ferenczi et al., 2017) and plant cells (Zaidi et al., 2017; Kim et al., 2017; Tang et al., 2017). The high efficiency and specificity of Cas12a in human cells, coupled with fewer off-target cleavage events compared to Cas9 (Kleinstiver et al., 2016), makes Cas12a a robust and reliable tool for genome editing.
For its in vitro characterization and crystallization (Swarts et al., 2017), Cas12a from Francisella novicida U112 was purified after heterologous expression in Escherichia coli. The expression strain E. coli RosettaTM 2 (DE3) carries a chromosomal T7 RNA polymerase gene under control of an IPTG inducible lacUV5 promoter. The cas12a gene is expressed using a pET vector (Studier and Moffatt, 1986; Rosenberg et al., 1987; Studier et al., 1990) with a lacI-controlled T7 promoter. Here we describe the steps required for controlled expression and purification of FnCas12a. The protocol can also be used for the expression and purification of Cas12a homologs from Acidaminococcus sp. and Lachnospiraceae bacterium.
Materials and Reagents
Note: Equivalent materials and reagents may be used as substitutes.
Expression of FnCas12a in E. coli RosettaTM 2(DE3)
100-ml Erlenmeyer flask (DWK Life Sciences, DURAN®, catalog number: 21 213 24 )
2-L Erlenmeyer flask (DWK Life Sciences, DURAN®, catalog number: 21 216 63 )
5-L Erlenmeyer flasks (DWK Life Sciences, DURAN®, catalog number: 21 216 73 )
50-ml conical centrifuge tubes (Sigma-Aldrich, catalog number: T2318-500EA )
2-ml screw top tube (Corning, catalog number: 430659 )
NalgeneTM PPCO Centrifuge Bottles with Sealing Closure (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3141-0500 ) or equivalent 500-ml centrifuge bottles
Pipette tips (DeckWorksTM standard pipet tips, Corning, catalog numbers: 4110 ; 4112 ; 4867 )
10-ml syringe (BD, catalog number: 309604 )
0.22 μm syringe filter (Mdi, catalog number: SYPL0601MNXX204 )
250-ml bottle (Greiner Bio One International, catalog number: 227261 )
Escherichia coli RosettaTM 2(DE3) cells (Merck, Novagen, catalog number: 71400 ) [encodes a T7 RNA polymerase gene under control of a lacUV5 promoter]
Plasmid pDS015* [pET His6 TEV LIC cloning vector (Addgene, catalog number: 29653 ), with F. novicida U112 cas12a gene insert fused to an N-terminal His-tag; expression under the control of a lacI-controlled T7 promoter]
*Note: Acidaminococcus sp. BV3L6 Cas12a (AsCas12a) and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) proteins can also be purified using this protocol with expression vectors 6His-MBP-TEV-huAsCpf1 (Addgene, catalog number: 90095 ) and 6His-MBP-TEV-huLbCpf1 (Addgene, catalog number: 90096 )
Tryptone (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0042B )
Yeast extract (BD, BactoTM, catalog number: 212720 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
Sodium hydroxide (NaOH) (Merck, EMD Millipore, catalog number: 106462 )
Ethanol (Fisher Scientific, catalog number: BP2818500 )
Chloramphenicol (Fisher Scientific, catalog number: BP904100 )
Kanamycin sulfate (Thermo Fisher Scientific, catalog number: 11815024 )
Glycerol (Fisher Scientific, catalog number: BP229-4 )
IPTG (Fisher Scientific, catalog number: BP1755-1 )
Agar (Acros Organics, catalog number: 400400050 )
LB medium (see Recipes)
1,000x chloramphenicol solution (34 mg/ml) (see Recipes)
1,000x kanamycin solution (50 mg/ml) (see Recipes)
1 M IPTG (IsoPropyl-1-Thio-β-D-Galactopyranoside) (see Recipes)
Glycerol stock (50% solution) (see Recipes)
Purification of FnCas12a
5 ml HisTrap HP (GE Healthcare, catalog number: 17524701 )
Dialysis tubing, high retention seamless cellulose tubing, avg. flat width 23 mm (0.9 in.), MWCO 12,400, 99.99% retention (Sigma-Aldrich, catalog number: D0405 )
Dialysis tubing clamps (Sigma-Aldrich, catalog number: Z371092 )
5 ml HiTrap Heparin HP (GE Healthcare, catalog number: 17040601 )
Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane (Merck, EMD Millipore, catalog number: UFC9100 )
HiLoad 16/600 Superdex 200 pg (GE Healthcare, catalog number: 28989335 )
GosselinTM Round-Base 10-ml Test Tubes (Corning, GosselinTM, catalog number: TP10-01 ) or other equivalent fraction collection tubes
NalgeneTM Oak Ridge High-Speed Centrifuge Tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3114-0050 ) or equivalent 50-ml centrifuge tubes
Membrane Filter, mixed cellulose esters (Merck, MF-Millipore, catalog number: HAWP04700 )
Membrane Filter, mixed cellulose esters (Merck, MF-Millipore, catalog number: GSWP04700 )
Cell pellet from overnight culture in which FnCas12a was expressed (from Procedure A)
cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, Roche Diagnostics, catalog number: 11873580001 )
Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876-5G )
β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
TEV protease (Sigma-Aldrich, catalog number: T4455 )
12% Mini-PROTEAN® TGXTM Precast Protein Gels (Bio-Rad Laboratories, catalog number: 4561043 )
4x Laemmli protein sample buffer for SDS-PAGE (Bio-Rad Laboratories, catalog number: 1610747 )
Bio-SafeTM Coomassie Stain (Bio-Rad Laboratories, catalog number: 1610786 )
PageRuler Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26619 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
Ethylenedinitrilotetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
Tris (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17926 )
Imidazole (Sigma-Aldrich, catalog number: I0250 )
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
Potassium chloride (KCl) (Merck, EMD Millipore, catalog number: 104933 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 757551 )
Glycine (Sigma-Aldrich, catalog number: G8898 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
1 M DTT (Dithiothreitol) stock (see Recipes)
0.5 M EDTA (Disodium Ethylene Diamine Tetra-Acetate) stock (pH 8) (see Recipes)
Lysis Buffer (see Recipes)
Wash Buffer (see Recipes)
Elution Buffer (see Recipes)
Dialysis Buffer (see Recipes)
Dilution Buffer (see Recipes)
IEX-A Buffer (see Recipes)
IEX-B Buffer (see Recipes)
SEC Buffer (see Recipes)
10x SDS-PAGE Electrophoresis Running Buffer (see Recipes)
Activity assay using purified Cas12a
Purified Cas12a Nuclease (from Procedure B)
Nuclease-free water
Proteinase K, Molecular Biology Grade (New England Biolabs, catalog number: P8107S )
crRNA containing the targeting sequence complementary to the target DNA
Note: The RNA can be ordered as a desalted RNA oligonucleotide or as PAGE-purified RNA oligonucleotide from an RNA synthesis company such as Sigma-Aldrich or IDT.
DNA substrate containing the target sequence and a 5’ TTTN PAM sequence
Note: The substrate DNA can be circular or linearized plasmid, PCR products, or synthesized oligonucleotides). As an example, the DNA substrate and crRNA used in the activity assay is shown in Figure 2.
Figure 2. Schematic of the Cas12a crRNA-DNA-targeting complex. The expected cleavage sites are indicated by red arrows.
GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0311 ) or equivalent
DNA gel Loading Dye [e.g., 6x DNA Loading Dye (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0611 )]
InvitrogenTM SYBRTM Safe DNA Gel Stain (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: S33102 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
Magnesium chloride hexahydrate (MgCl2·6H2O)
HEPES (Sigma-Aldrich, catalog number: H3375 )
Ethylenedinitrilotetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
10x Nuclease Reaction Buffer (see Recipes)
Equipment
Note: Equivalent equipment can be used.
Expression of FnCas12a in E. coli RosettaTM 2
Pipettes (Corning, model: LambdaTM Plus Single-Channel Pipettor, catalog numbers: 4070 ; 4074 ; 4075 )
New BrunswickTM Innova® 42 incubator (Eppendorf, New BrunswickTM, model: Innova® 42 , catalog number: M1335-0002) or an equivalent incubator that can be set at 37 °C
Sorvall LYNX 4000 Superspeed Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall LYNX 4000 , catalog number: 75006580) or an equivalent centrifuge that can be cooled down to 4 °C and can perform up to 6,000 x g
New BrunswickTM Innova® 44/44R (Eppendorf, New BrunswickTM, model: Innova® 44/44R , catalog number: M1282-0002) or any equivalent shaker incubator where the temperature can be set at 37 °C and 18 °C
Cell density meter (GE Healthcare, model: UltrospecTM 10 , catalog number: 80-2116-30), or equivalent spectrophotometer that can measure the density of cells in suspension at 600 nm
Ice-water bath (water and ice mixed)
Purification of FnCas12a
SONOPULS HD (Bandelin electronic, model: HD 3200 ) with VS 70 T Sonotrode (Bandelin) or equivalent ultrasonic homogenizer/Sonifier, or alternatively a French Pressure Cell (French Press) for cell lysis
Peristaltic pump P-1 with connectors for 5 ml HisTrap HP (GE Healthcare, model: Peristaltic Pump P-1, catalog number: 18111091 ) or an equivalent peristaltic pump
Tubing Connectors for Use with Peristaltic Pump P-1 (GE Healthcare, catalog number: 11300082 )
ÄKTApurifier 10 FPLC system (GE Healthcare, model: ÄKTApurifier 10 , catalog number: 28406264) or an equivalent FPLC system
Sorvall LYNX 4000 Superspeed Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall LYNX 4000 , catalog number: 75006580) or an equivalent centrifuge that can be cooled down to 4 °C and can perform up to 30,000 x g
pH meter (QiS, model: B210 )
Filter holder assembly for filtration (Merck, catalog number: XX1014700 or Nalgene, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: DS0320-2545 ), or equivalent filter holder assembly
Diaphragm Vacuum Pumps LABOPORT® N 820 (ABM van Zijl B.V, catalog number: ABMK N8203FT18 ), or an equivalent vacuum pump
Nanodrop (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 , catalog number: ND-2000)
Mini-PROTEAN Tetra cell (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell, catalog number: 1658004EDU ), or an equivalent vertical electrophoresis system
Epson Perfection V850 Pro scanner (Epson, model: Perfection V850 Pro ) or equivalent scanner or imager suitable for SDS-PAGE gel imaging.
Activity assay using purified Cas12a
EppendorfTM 5424 Microcentrifuge (Eppendorf, model: 5424 , catalog number: 022620498)
MUPID One Horizontal Electrophoresis System (Bulldog Bio, catalog number: MU2 ) or an equivalent horizontal electrophoresis system
G:BOX F3 (Syngene, model: G:BOX F3 , catalog number: 05-GBOX-F3) gel doc system or equivalent DNA agarose gel imaging equipment
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mohanraju, P., Oost, J. V. D., Jinek, M. and Swarts, D. C. (2018). Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein. Bio-protocol 8(9): e2842. DOI: 10.21769/BioProtoc.2842.
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Category
Microbiology > Heterologous expression system > Escherichia coli
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Isolation and purification
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2,843 | https://bio-protocol.org/exchange/protocoldetail?id=2843&type=1 | # Bio-Protocol Content
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Morphological Analysis of Dopaminergic Neurons with Age Using Caenorhabditis elegans GFP Reporter Strains
Sanjib Guha
GC Guy Caldwell
Pankaj Kapahi
Published: May 5, 2018
DOI: 10.21769/BioProtoc.2843 Views: 6269
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Abstract
This protocol describes how to quantify different morphological defects as observed in the dopaminergic neurons using C. elegans GFP reporter strains with age.
Keywords: Dopaminergic neurons Neurodegeneration Aging GFP Reporter strains C. elegans
Materials and Reagents
Plastic plates , 60 x 15 mm (Olympus Plastics, catalog number: 32-105G )
Microscopic slides (Denville Scientific, catalog number: M1002 )
Micro cover glass, 22 x 22 mm (VWR, catalog number: 48366-067 )
OP50-1 Bacteria (E. coli https://cgc.umn.edu/strain/OP50-1)
C. elegans reporter strains
BY200 - Pdat-1::GFP(vtIs1) V (Michael Aschner, Albert Einstein College of Medicine, NY, USA)
BY250 - Pdat-1::GFP(vtIs7) V (Randy Blakely, Florida Atlantic University, FL, USA)
UA44 - [baInl1; Pdat-1::α-syn high, Pdat-1::gfp] (Guy Caldwell, University of Alabama, AL, USA)
UA57 - baIs4 [dat-1p::GFP + dat-1p::CAT-2] (CGC, University of Minnesota, MN, USA)
Sodium hypochlorite solution (Avantor Performance Materials, J.T. Baker, catalog number: 9416-01 )
M9 buffer
NGM agar
Agarose LE (Apex Chemicals and Reagents, catalog number: 01132-34 )
Levamisole hydrochloride (MedChemExpress, catalog number: HY-13666 )
Immersion Oil (Immersol 518F, Carl Zeiss, catalog number: 444960-0000-000 )
Equipment
Centrifuge
Shaker
Fluorescent Microscope (Olympus, model: BX51 , TRF)
Digital camera (Hamamatsu ORCA-ER, version: C4742-80)
Software
Image J
GraphPad Prism
Procedure
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Category
Neuroscience > Development > Neuron
Cell Biology > Tissue analysis > Tissue imaging
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2,844 | https://bio-protocol.org/exchange/protocoldetail?id=2844&type=0 | # Bio-Protocol Content
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Quantification of Salicylic Acid (SA) and SA-glucosides in Arabidopsis thaliana
VA Valérie Allasia
BI Benoit Industri
MP Michel Ponchet
MQ Michaël Quentin
Bruno Favery
HK Harald Keller
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2844 Views: 11860
Edited by: Marisa Rosa
Reviewed by: John V. DeanRekha R. Warrier
Original Research Article:
The authors used this protocol in Apr 2016
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The authors used this protocol in:
Apr 2016
Abstract
Homeostasis between the cytoplasmic plant hormone salicylic acid (SA) and its’ inactive, vacuolar storage forms, SA-2-O-β-D-glucoside (SAG) and SA-β-D-Glucose Ester (SGE), regulates the fine-tuning of defense responses to biotrophic pathogens in Arabidopsis thaliana. This protocol describes a simplified, optimized procedure to extract and quantify free SA and total hydrolyzable SA in plant tissues using a classical HPLC-based method.
Keywords: Salicylic acid SA-glucoside Defense hormone Arabidopsis thaliana HPLC
Background
SA (2-hydroxybenzoic acid) is a plant hormone, which is synthesized in the chloroplast in response to pathogen attack. It is then exported to the cytoplasm, where it establishes both local and systemic-acquired resistance (SAR). In a generalized scheme, plant resistance to biotrophic pathogens is thought to be mediated through SA signaling, whereas resistance to necrotrophic pathogens is controlled by jasmonic acid (JA) and ethylene (ET). SA and JA/ET signaling pathways interact antagonistically. SA accumulation to high concentrations is toxic and leads to cell- and tissue damage. Most pathogen-induced SA is thus glycosylated by UDP-glucosyltransferases (UGTs) to form hydrophilic, non-toxic SAG and SGE (Noutoshi et al., 2012; George Thompson et al., 2017). SAG and SGE are then sequestered in vacuoles, where they form reusable sources for hydrolysis to active SA. Increasing amounts of total SA (SA + SAG/SGE) in plant tissues thus reflect SA synthesis as a response to biotrophic pathogen attack. However, the amplitude of defense responses in infected plant tissues is determined by the amount of available cytoplasmic, unconjugated SA. To evaluate both the onset of SA-dependent defense responses and their amplitude, it is essential to quantify free and conjugated SA, respectively. This article describes a method for measuring conjugated and unconjugated SA levels in phase-partitioned extracts from A. thaliana seedlings. It is based on a protocol established for SA analysis in cucumber leaves (Meuwly and Métraux, 1993), which we optimized and downscaled for convenient, routine use.
Materials and Reagents
Pipette tips
Nitrile gloves
Microcentrifuge tubes (2 ml) (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 69720 )
Centrifuge tubes (15 ml) (e.g., Corning, catalog number: 430791 )
Microcentrifuge Tube Locks (LidLocksTM, VWR, catalog number: 14229-941)
Manufacturer: Sorenson Bioscience, catalog number: 11870 .
Glass wool (e.g., Sigma-Aldrich, catalog number: 18421 )
Ten-day-old Arabidopsis thaliana seedlings (e.g., different genetic wild-type or mutant backgrounds, inoculated with pathogen or otherwise treated)
Liquid nitrogen
Ethanol (EtOH; e.g., Sigma-Aldrich, catalog number: 24103 )
70% aqueous EtOH (v/v)
Methanol HPLC grade (MeOH; e.g., CARLO ERBA Reagents, catalog number: 412383 )
90% aqueous MeOH (v/v)
Trichloracetic acid (TCA; e.g., Sigma-Aldrich, catalog number: T9159 )
20% aqueous TCA (w/v)
Ethyl acetate, analytical grade (e.g., CARLO ERBA Reagents, catalog number: 448256 )
Cyclohexane, analytical grade (e.g., CARLO ERBA Reagents, catalog number: 436903 )
A mixture of ethyl acetate and cyclohexane (1:1, v:v)
Trifluoracetic acid (TFA; e.g., Sigma-Aldrich, catalog number: T62200 )
10% aqueous MeOH (v/v) with 0.1% TFA (v/v)
82% aqueous MeOH (v/v) with 0.1% TFA (v/v)
Concentrated hydrochloric acid (HCl; 37%, 12 M; e.g., Sigma-Aldrich, catalog number: 30721 )
Ultra-pure water
2-Methoxybenzoic acid, o-Anisic acid (OAA; e.g., Sigma-Aldrich, catalog number: 169978 )
Sodium salicylate (e.g., Sigma-Aldrich, catalog number: S3007 )
o-Anisic acid 50x stock solution (see Recipes)
Equipment
Micropipettes (e.g., Gilson, model: P2 , P20 , P200 , P1000 )
Mortar (40 ml content) with pestle
Fume hood
Water purification system (Merck, EMD Millipore, catalog number: SYNS0HFWW )
Vortex (e.g., IKA, model: MS 1 minishaker )
Dry bath heating block for 2 ml microcentrifuge tubes (e.g., Major Science, model: EL-02 )
Vacuum concentrator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Savant SpeedVac Concentrator )
Vacuum pump (e.g., BÜCHI Labortechnik, model: V-300 )
Microcentrifuge (e.g., Eppendorf, model: 5415 D with Rotor: Eppendorf, model: F45-24-11 )
C18 HPLC column (e.g., Inertsil 5 ODS3, 5 µm, 250 x 4.6 mm i.d., Interchim, France) (GL Sciences, model: Inertsil® ODS3 )
HPLC System (Shimadzu Prominence LC System) equipped with:
2 solvent delivery units (Shimadzu Scientific, model: LC-20AD )
A system controller (Shimadzu Scientific, model: CBM-20A )
An autosampler (Shimadzu Scientific, model: SIL-20AC )
A column oven (Shimadzu Scientific, model: CTO-20A )
A diode array detector (Shimadzu Scientific, model: SPD-M20A )
A fluorescence detector (Shimadzu Scientific, model: RF-10AXL )
Software
Chromatography data system software (e.g., WATERS, Empower 3 Pro Chromatography Data Software)
Microsoft Excel
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Allasia, V., Industri, B., Ponchet, M., Quentin, M., Favery, B. and Keller, H. (2018). Quantification of Salicylic Acid (SA) and SA-glucosides in Arabidopsis thaliana. Bio-protocol 8(10): e2844. DOI: 10.21769/BioProtoc.2844.
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Category
Plant Science > Plant biochemistry > Plant hormone
Plant Science > Plant immunity > Host-microbe interactions
Biochemistry > Other compound > Plant hormone
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2,845 | https://bio-protocol.org/exchange/protocoldetail?id=2845&type=0 | # Bio-Protocol Content
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In vitro Analysis of Ubiquitin-like Protein Modification in Archaea
Xian Fu
ZA Zachary Adams
Julie A. Maupin-Furlow
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2845 Views: 5827
Edited by: Elizabeth Libby
Reviewed by: Srujana Samhita YadavalliAgnès Groisillier
Original Research Article:
The authors used this protocol in Sep 2017
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The authors used this protocol in:
Sep 2017
Abstract
The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii, a halophilic archaeon serving as the model organism.
Keywords: Archaea Ubiquitin (Ub) Post-translational modification SAMP
Background
The process by which ubiquitin (Ub) is covalently attached to target proteins is termed ubiquitination, which controls an enormous range of cellular process in eukaryotic cells (Glickman and Ciechanover, 2002; Komander and Rape, 2012). Ubiquitination is catalyzed by a cascade of enzymes including an Ub-activating enzyme (E1), Ub-conjugating enzymes (E2s), and Ub ligases (E3s). In vitro reconstitution of ubiquitination is a useful assay to determine the specificity between enzymes or between E3s and protein substrates (Zhao et al., 2012). In Archaea, the Ubl protein SAMP adopts a Ub-fold and is isopeptide-linked to protein targets catalyzed by an E1-like enzyme UbaA [reviewed in Maupin-Furlow, (2014)]. While E1 homologs are widespread in Archaea, canonical E2 or E3 enzymes are not predicted in most Archaea based on primary sequence comparisons. Our recent study of Haloferax volcanii, shows methionine sulfoxide reductase A (MsrA) is needed for Ubl protein modification (sampylation) together with UbaA under a mild oxidative condition in vivo and in vitro (Fu et al., 2017). Here, we describe a detailed in vitro protocol to reconstitute and analyze MsrA-dependent sampylation.
Materials and Reagents
Gloves (Fisherbrand, Fisher Scientific, catalog number: 19-130-1597C )
Sterile toothpicks (Royal Paper Products, Inc., item number: R820)
Ampac 500 series SealPAK heavy duty pouches (10.2 x 15.2 cm) (Fisher Scientific, catalog number: 01-812-25D) (seal Western blot membrane with CDP-Star or ECL prime reagents in pouches using colored labeling tape)
Manufacturer: Ampac, catalog number: 50024 .
Sterile loop (Fisherbrand, Fisher Scientific, catalog number: 22-363-599 )
Fernbach flasks (2.8 L, wide mouth) (Corning, PYREX®, catalog number: 4420-2XL )
Baffled culture flasks (250 ml) (DWK Life Sciences, Kimble, catalog number: 25630-250 )
Aluminum foil (Fisherbrand, Fisher Scientific, catalog number: 01-213-105 ) (used to wrap items for autoclaving)
Sterile polystyrene disposable serological 10 ml pipets with magnifier stripe (Fisherbrand, Fisher Scientific, catalog number: 13-678-11E )
Polypropylene round-bottom centrifuge tubes, 50 ml (Nalgene, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3119-0050 , item number: UX-06327-47)
Nalgene rapid-flow sterile disposable bottle top 0.2 µm filters with surfactant-free cellulose acetate (SFCA) membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 290-3320 )
HisTrap HP column (5 ml) (GE Healthcare, catalog number: 17524802 )
StrepTrap HP column (1 ml) (GE Healthcare, catalog number: 29048653 )
13 x 100 mm2 culture tubes (Fisher Scientific, catalog number: 14-961-27 )
Borosilicate glass tubes with plain end (13 x 100 mm) (Fisherbrand, Fisher Scientific, catalog number: 14-961-27 ) (used for cell culture)
Amicon Ultra-4 centrifugal filter unit with Ultracel-3 membrane (NMWL 3 kDa) (Merck, catalog number: UFC800308 )
Superdex 75 10/300 GL column (GE Healthcare, catalog number: 17517401 )
Surfactant-free cellulose acetate (SFCA) membrane syringe filters (28-mm membrane with 0.2, 0.45 and 0.8 µm pore sizes in acrylic housing) (Corning, catalog numbers: 431219 , 431220 , and 431221 )
SnakeSkin dialysis tubing (3.5K MWCO, 22 mm I.D.) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 68035 )
1.5 ml microcentrifuge tubes (Fisherbrand, Fisher Scientific, catalog number: 02-681-320 )
2.0 ml microcentrifuge tubes (Fisherbrand, Fisher Scientific, catalog number: 02-681-321 )
Zeba Spin Desalting Columns (7K MWCO) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 89882 )
Amersham Hybond P 0.45 polyvinylidene difluoride (PVDF) membrane (GE Healthcare, catalog number: 10600023 )
Disposable plastic cuvettes (Fisherbrand, Fisher Scientific, catalog number: 149-551-27 )
X-ray film (RPI, catalog number: 248300 )
Rack LTS tips (‘P20’ 2-20 µl, ‘P200’ 20-200 µl, ‘P1000’ 100-1,000 µl) (Mettler-Toledo, Rainin, catalog numbers: 17001865 , 17001863 and 17001864 )
10 ml Luer-Lok syringe (BD, catalog number: 309695 )
Kimwipes Delicate Task Wipers, 1-Ply (KWCC, Kimberly-Clark, catalog number: 34155 )
Colored labeling tape, rainbow pack (Fisherbrand, Fisher Scientific, catalog number: 15-901-10R )
Four square cassettes (8 x 10") (Fisher Scientific, catalog number: FBXC-810 ) (for exposure of Western blot membrane to X-ray film)
Glass storage/media bottles (DWK Life Sciences, Kimble, catalog numbers: 14395-500 for 500 ml and 14395-1000 for 1,000 ml)
Polypropylene griffin low-form plastic beakers (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 1201-4000 for 2,000 ml and 1201-1000 for 1,000 ml)
Polyvinyl wrapping film (Fisherbrand, Fisher Scientific, catalog number: 15-610 ) (can store SDS-PAGE gels wrapped in Kimwipes that are moistened with deionized water and further wrapped with polyvinyl film for up to 2 weeks at 4 °C prior to electrophoresis)
Polystyrene cuvettes (1.5 ml capacity) (Fisherbrand, Fisher Scientific, catalog number: 14-955-127 ) (used for assays in visible spectral range, 340 to 750 nm)
Polypropylene plastic graduated cylinders (Nalgene, Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 3664-0050 , 3664-0250 , and 3664-1000 )
Polypropylene closures (DWK Life Sciences, Kimble, catalog number: 73660-13 ) (used with 13 mm O.D. plain-end culture tubes)
Petri dishes with clear lid (Fisherbrand, Fisher Scientific, catalog number: FB0875712 )
Cells
Haloferax volcanii LR03 (Δsamp1 Δsamp2 Δsamp3 ΔmsrA ΔubaA) carrying plasmid pJAM3010 (a plasmid encoding msrA-strepII under control of the P2rrnA constitutive promoter) (Fu et al., 2017) or pJAM1209 (a plasmid encoding his6-ubaA under control of the P2rrnA constitutive promoter) (Hepowit et al., 2016) or without carrying any plasmid
Note: H. volcanii strains and plasmids are available upon request.
Escherichia coli Rosetta (DE3) (Novagen, Merck, catalog number: 70954-3 ) carrying plasmid pJAM3200 (a plasmid encoding msrA-strepII under control of T7 promoter) from the Maupin-Furlow lab (Fu et al., 2017) or plasmid pJAM1132 (a plasmid encoding flag-his6-samp2 under control of T7 promoter) from the Maupin-Furlow lab (Hepowit et al., 2016)
Note: Plasmids are available from the Maupin-Furlow lab upon request.
MsrA-StrepII (purified as per procedure outlined below)
His6-UbaA (purified as per procedure outlined below)
Deionized H2O (for details see Equipment item number 27)
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Novobiocin (Sigma-Aldrich, catalog number: N1628 )
Kanamycin sulfate (Fisher BioReagents, Fisher Scientific, catalog number: BP906-5 )
Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fisher BioReagents, Fisher Scientific, catalog number: BP1620-1 )
d-Desthiobiotin (Sigma-Aldrich, catalog number: D1411-500MG )
Imidazole, ACS Reagent, ≥ 99% titration (Sigma-Aldrich, catalog number: I2399 )
Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, catalog number: 1610436 )
Pierce Bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
Albumin, bovine (BSA) (lyophilized powder, ≥ 96% purity by agarose gel electrophoresis) (Sigma-Aldrich, catalog number: A2153 )
Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Chemical, Fisher Scientific, catalog number: M35-12 )
Bortezomib proteasome inhibitor (free base, > 99% purity) (LC Laboratories, catalog number: B-1408 )
DNase I from bovine pancreas (Sigma-Aldrich, catalog number: D4263-1VL ), a standardized vial containing 2,000 Kunitz units of DNase I (Sigma-Aldrich, catalog number: D4527 ) at ≥ 0.25 mg total protein
Dimethylformamide (DMF) (Fisher BioReagents, Fisher Scientific, catalog number: BP1160-500 )
Sodium chloride (NaCl) (Fisher Chemical, Fisher Scientific, catalog number: S642-12 )
Adenosine 5’-triphosphate (ATP), disodium salt hydrate, 98% (ACROS Organics, catalog number: 102800100 )
Dimethyl sulfoxide (DMSO), molecular biology grade (Sigma-Aldrich, catalog number: D8418-50ML ) (for in vitro assays)
DMSO, Certified ACS (Fisher Chemical, Fisher Scientific, catalog number: D128-1 ) (for culturing cells)
Dithiothreitol (DTT) (Fisher BioReagents, Fisher Scientific, catalog number: BP172-5 )
Antibodies
Strep-tag II monoclonal antibody (in mouse) (QIAGEN, catalog number: 34850 )
Anti-His IgG2 monoclonal antibody (in mouse) (GE Healthcare, catalog number: 27471001 )
HRP-conjugated His6 monoclonal antibody (Proteintech, catalog number: HRP-66005 ) (anti-His6 antibody replaced the discontinued GE Healthcare product listed above)
Goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (Sigma-Aldrich, catalog number: A5153-1ML )
Alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (Sigma-Aldrich, catalog number: A9469-2MG )
Nonfat dry milk (instant, powdered) (Publix, Lakeland, FL item)
Tropix CDP-Star chemiluminescent substrate (12.5 mM concentrate) (Applied BioSystems, Thermo Fisher Scientific, InvitrogenTM, catalog number: T2304 )
cOmplete His-tag purification resin (Sigma-Aldrich, Roche Diagnostics, catalog number: 5893682001 )
Sodium phosphate monobasic monohydrate (H2NaO4P·H2O) (Fisher Chemical, Fisher Scientific, catalog number: S369-1 )
Potassium phosphate monobasic (KH2PO4) (Fisher Chemical, Fisher Scientific, catalog number: P285-500 )
Ethylenediaminetetraacetic acid (EDTA) (Fisher BioReagents, Fisher Scientific, catalog number: BP120-500 )
EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, Roche Diagnostics, catalog number: 11873580001 )
SYPRO Ruby protein gel stain (Bio-Rad Laboratories, catalog number: 1703125 )
Bio-Safe Coomassie stain (Bio-Rad Laboratories, catalog number: 1610786 )
Potassium sulfate (K2SO4) (Fisher Chemical, Fisher Scientific, catalog number: P304-3 )
Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Chemical, Fisher Scientific, catalog number: C79-500 )
Bacto dehydrated culture media additive: Tryptone (BD, BactoTM, catalog number: 211705 )
BBL dehydrated culture media additive: Yeast extract (BD, BBLTM, catalog number: 211929 )
Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-212 )
Agar (Sigma-Aldrich, catalog number: A7002 )
Tris-base (Fisher BioReagents, Fisher Scientific, catalog number: BP152-1 )
Concentrated HCl (Fisher Chemical, Fisher Scientific, catalog number: A481-212 )
Sodium dodecyl sulfate (SDS) (Fisher BioReagents, Fisher Scientific, catalog number: BP166-500 )
Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
Acrylamide/bis-acrylamide (37.5:1, 40%) (electrophoresis grade) (Fisher BioReagents, Fisher Scientific, catalog number: BP1410-1 )
Tetramethylethylenediamine (TEMED, electrophoresis grade) (Fisher BioReagents, Fisher Scientific, catalog number: BP150-100 )
Ammonium persulfate (APS) (Bio-Rad Laboratories, catalog number: 1610700 )
2-(N-Morpholino)ethanesulfonic acid (MES) hydrate (ACROS Organics, catalog number: 172595000 )
Methanol (Fisher Chemical, Fisher Scientific, catalog number: A413-4 )
Tween 20 (molecular biology grade) (Sigma-Aldrich, catalog number: P9416 )
Glycine (Bio-Rad Laboratories, catalog number: 1610718 )
Amersham ECL prime Western blotting detection reagent (ECL Prime) (GE Healthcare, catalog number: RPN2232 )
Precision plus protein dual color standards (500 µl) (Bio-Rad Laboratories, catalog number: 1610374 )
ATCC974 medium (see Recipe 1)
LB medium (see Recipe 2)
Lysis buffer for HisTrap HP chromatography (see Recipe 3)
Lysis buffer for StrepTrap HP chromatography (see Recipe 4)
Tris-salt buffer (see Recipe 5)
Concentrated assay buffer (see Recipe 6)
2x SDS reducing buffer (see Recipe 7)
12% SDS-PAGE gels (see Recipe 8)
10x running buffer (see Recipe 9)
Transblot buffer (see Recipe 10)
10x TBS (see Recipe 11)
Tris-buffered saline with 0.1% Tween 20 (TBST) (see Recipe 12)
Equipment
Sorvall Evolution RC centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Evolution RC, catalog number: 728211 ) (used to centrifuge samples at 4 to 15 °C and ≤ 12,000 x g)
Fiberlite F9-4x 1000y fixed angle superspeed rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 76981 ) and Sorvall SS-34 fixed angle rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28020 ) (used with Sorvall Evolution RC to centrifuge 1 L cultures and 50 ml cell extract, respectively)
French Press G-M (Glen Mills, model: Model 11, catalog number: 5500-000011 ) (used for lysis of cells at 20,000 to 24,000 psi)
French Press standard pressure cell (35 ml, 40,000 psi) (Glen Mills, catalog number: 6800-FA-032 ) (used with French Press, item 3)
Sorvall RC-3 general purpose centrifuge (Newton, CT) (used for centrifugal filtration with an HL-8 swinging bucket rotor at 4 °C and 4,000 x g)
Water bath (LAUDA-Brinkmann, model: LAUDA Aqualine AL 12 , catalog number: L000610) (used to incubate reactions at 45 °C)
Two bench-top microcentrifuges (Eppendorf, model number: 5418 , catalog number: 022620304) (used for centrifugation of 1-2 ml samples at up to 10,000 x g) (place one centrifuge in the cold room for 4 °C and the other on the bench-top for room temperature)
BenchRocker 2D rocker (Alkali Scientific, catalog number: RS7235 )
Autoclave (Consolidated Sterilizer Systems, model: SR-24C )
Mini-PROTEAN Tetra Handcast Systems (Bio-Rad Laboratories, catalog number: 1658005 )
SmartSpec 3000 Plus spectrophotometer (Bio-Rad Laboratories, catalog number: 1702525 ) (use for BCA assay at A562 nm and monitoring of growth at OD600)
PowerPac Basic 300 V power supply (Bio-Rad Laboratories, catalog number: 1645050 ) (used for transblot overnight at 20 V and SDS-PAGE for up to 90 min at 150 V)
New Brunswick I24 shaker, 3/4" orbit (7-60 °C) (Eppendorf, New BrunswickTM, model: I24 , catalog number: M1344-0000) (used for culturing cells at 37 to 42 °C at 200 rpm)
Refrigerator (4 °C) (Frigidare, model: FRU17B2JW )
Puffer Hubbard (-20 °C) (Revco Tech, model: 1UF1821A14 )
Ultra-low temperature freezer (-80 °C) (Eppendorf, New BrunswickTM, model: C660-86 )
XP-Series toploading balance (Denver Instrument, model: XP-600 )
Analytical balance (Mettler Toledo, model: B balance line, AB54 )
Vortex mixer (Thermolyne, catalog number: 37600 )
Chemical fume hood (St. Charles Manufacturing, St. Charles, IL)
Pipettes (2-20 µl, 20-200 µl, 100-1,000 µl) (Rainin type LTS)
pH meter (Corning, model: Model 320) (used to titrate buffers to pH 6.8 to 8.8)
Scanner (Epson Perfection, model: 3170 Photo ) (used for scanning exposed X-ray film and Coomassie Blue R250 stained protein gels at 300-600 dpi)
Mini trans-blot module (Bio-Rad Laboratories, catalog number: 1703935EDU )
Konica X-ray film processor (Konishiroku Photo Industry, model: QX60A )
Electrophoresis systems autoradiography cassette (Fisher Scientific, catalog number: FBXC-810 )
Siemens Vantage Reverse Osmosis Systems (Siemens, model: M21 series, model number: M21R004EA ) with EVOQUA filters (Siemens, catalog number: C1207098 ) and Atlantic Ultraviolet Germicidal UV Equipment (Atlantic Ultraviolet, model: MP49 ) (use water purification system to generate deionized water)
BioLogic DuoFlow 10 System (Bio-Rad Laboratories, catalog number: 7600037 ) (used for protein chromatography by step gradient at flow rates of 0.5 to 1.2 ml∙min-1 and monitoring of protein fractions by A280)
Stirring hotplate (PC-220 Pyroceram) (Corning, catalog number: 6795-220 ) used with magnetic stir bars (Octagonal Magnetic Stir Bar Kit) (Fisherbrand, Fisher Scientific, catalog number: 14-513-82 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Fu, X., Adams, Z. and Maupin-Furlow, J. A. (2018). In vitro Analysis of Ubiquitin-like Protein Modification in Archaea. Bio-protocol 8(10): e2845. DOI: 10.21769/BioProtoc.2845.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Activity
Biochemistry > Protein > Isolation and purification
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2,846 | https://bio-protocol.org/exchange/protocoldetail?id=2846&type=0 | # Bio-Protocol Content
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Ex vivo Follicle Rupture and in situ Zymography in Drosophila
EK Elizabeth M Knapp
LD Lylah D Deady
JS Jianjun Sun
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2846 Views: 6388
Edited by: Yanjie Li
Original Research Article:
The authors used this protocol in Dec 2017
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Original research article
The authors used this protocol in:
Dec 2017
Abstract
Ovulation, the process of releasing a mature oocyte from the ovary, is crucial for animal reproduction. In order for the process of ovulation to occur, a follicle must be fully matured and signaled to rupture from the ovary. During follicle rupture in both mammals and Drosophila, somatic follicle cells are enzymatically degraded to allow the oocyte to be liberated from the follicle. Here, we describe a detailed protocol of our newly developed ex vivo follicle rupture assay in Drosophila, which represents a first assay allowing direct quantification of follicles’ capacity to respond to ovulation stimuli and rupture. This assay can be modified to stimulate rupture with other reagents (for example, ionomycin) or to query enzymatic activity (in situ zymography). In addition, this assay allows genetic or pharmacological screens to identify genes or small molecules regulating follicle rupture in Drosophila.
Keywords: Drosophila Ovulation Follicle rupture Octopamine in situ zymography Follicle cells
Background
The study of ovulation in Drosophila has largely been limited due to technical challenges in direct visualization and quantification of ovulation events. In the last several decades, multiple indirect methods have been developed with limitations. The first method for the study of ovulation in Drosophila is to push the female’s abdomen and record if an egg was ejected from the ovipositor (Aigaki et al., 1991; Lee et al., 2003). This method is a rough estimate of whether there’s an ovulated egg inside the uterus. The second common assay is to measure the egg-laying capacity of female flies after mating. The egg-laying process is complex, involving development and maturation of a follicle, ovulation, transportation of the ovulated egg through the oviduct, selection of an appropriate egg-laying substrate, and oviposition (Spradling, 1993; Bloch Qazi et al., 2003). If any of these processes are impaired, it will result in defective egg laying. Another indicator that ovulation is impaired is an egg-retention phenotype (Monastirioti et al., 1996; Monastirioti, 2003; Cole et al., 2005). If a female has an excess of mature follicles within her ovaries, it indicates an anovulatory phenotype. However, this can be caused directly by an ovulation defect or indirectly by a defect downstream of ovulation in the egg-laying process. On the other hand, a lack of egg-retention phenotype does not necessarily mean a lack of ovulation defect. A fourth type of assay used to study ovulation is to examine if an egg is present in the reproductive tract (Heifetz et al., 2000; Lee et al., 2009; Lim et al., 2014). Variations in this assay range from quantifying ovulation rate by the percentage of females with an egg in their lower oviduct/common oviduct/uterus post mating (Lee et al., 2009; Lim et al., 2014), to examining the distribution of eggs in each of these separate regions over time (Heifetz et al., 2000). However, each of these assays could be influenced by the speed of oogenesis, ovulation, and oviposition. To account for all the possible drawbacks of each individual assay, we recently combined the egg-retention assay, the egg-laying assay, and the egg location in the female reproductive tract to estimate the average time for ovulating an egg (ovulation time; Sun and Spradling, 2013; Deady et al., 2015 and 2017; Deady and Sun, 2015; Knapp and Sun, 2017). However, this method is tedious and also relies on the indirect measurements of ovulation.
We recently characterized ovulation at a cellular level and discovered that Drosophila ovulation involves a follicle rupture process. During ovulation, posterior follicle cells activate matrix metalloproteinase 2 (Mmp2), which degrades posterior follicle cells allowing for the encased oocyte to rupture into the oviduct (Deady et al., 2015). We also found that this process is initiated by direct octopamine (OA) and octopamine receptor in mushroom body (Oamb) signaling in follicle cells, and the entire process can be recapitulated in our ex vivo culture system (Deady and Sun, 2015). We named this assay ex vivo follicle rupture, in which mature follicles are isolated from the ovary and stimulated with OA to induce follicle rupture. Percent of follicles ruptured can be reported at the end of a short three-hour incubation, which is a direct quantification of follicle rupture. This assay allows for a relatively simple, high-throughput examination of follicle rupture in Drosophila, and is ideal for genetic and pharmacological screens. However, this experiment is done ex vivo, and results should be verified in vivo using some of the assays described above.
Materials and Reagents
Utility boxes, 500 ml, Nalgene (Thermo Fisher ScientificTM, catalog number: 5700-0500 )
Aluminum foil
Paper towel
PYREXTM spot plates (9-well) (Corning, PYREX®, catalog number: 7220-85 )
Stainless steel needles, 0.25 mm, 36 mm (Ted Pella, catalog number: 13561 )
Plastic transfer pipets, disposable, 5.8 ml (Fisher Scientific, Fisherbrand, catalog number: 13-711-9CM )
1.5 ml Eppendorf tubes
Grace’s Media, with L-Glutamine (Genesee Scientific, catalog number: 25-516G )
Dry yeast, active (Genesee Scientific, catalog number: 62-103 )
Cornmeal (Genesee Scientific, catalog number: 62-101 )
Molasses (Fisher Scientific, catalog number: NC9109740)
Manufacturer: LabScientific, catalog number: FLY-8008-16 .
Agar (Genesee Scientific, catalog number: 66-103 )
Tegosept (Genesee Scientific, catalog number: 20-258 )
Ethanol
Propionic acid (Sigma-Aldrich, catalog number: P1386-1L )
Fetal bovine serum (Atlanta Biologicals, catalog number: S11150 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Octopamine hydrochloride (Sigma-Aldrich, catalog number: O0250-1G )
Fluorescein-conjugated DQTM Gelatin From Pig Skin (Thermo Fisher Scientific, InvitrogenTM, catalog number: D12054 )
Wet yeast paste (see Recipes)
Fly food (see Recipes)
Culture media mixture (see Recipes)
Octopamine stock solution (see Recipes)
Octopamine working solution (see Recipes)
Fluorescein-conjugated DQTM Gelatin stock solution (see Recipes)
Gelatin working solution (see Recipes)
Equipment
Dumont #5 forceps (Fine Science Tools, catalog number: 11252-20 )
Pipetman kit G series (P20, P200, P1000) (Gilson, catalog number: F167900 )
Modular stereo microscope for fluorescent imaging (Leica Microsystems, model: Leica MZ10 F )
sCMOS camera (PCO. 4.2)
29 °C incubator with humidity control–set to ~70% RH (Percival Scientific, model: DR-36VL )
CO2 tank, CD 50 (Airgas, model: CGA-320 )
Autoclave
Flypad (Genesee Scientific, catalog number: 59-114 )
Nutator (Fisher Scientific, model: 260100F )
Software
ImageJ (NIH) (Schneider et al., 2012)
Micro-Manager (NIH) (Schneider et al., 2012; Edelstein et al., 2014)
Procedure
Fly rearing
Genetic requirements
A fluorescent reporter expressed in mature follicle cells (stage 14) is required for isolating mature follicles with an intact follicle-cell layer. In our experiments, we used either R47A04-Gal4 or R44E10-Gal4 from the Janelia Gal4 collection (Pfeiffer et al., 2008) to drive the expression of a UAS-RFP reporter specifically in stage-14 follicle cells.
Collect virgin females with correct genotype and age them for 5-6D at 29 °C.
Supplement food with ~1 teaspoon of fresh yeast paste 2-3D before the experiment.
Experimental preparation
Warm Grace’s media to room temperature (~25 °C).
Prepare the culture media mixture (see Recipes).
Line utility boxes with aluminum foil to shield inside from the light.
Add a damp paper towel to the bottom of the box to maintain moisture during the three-hour incubation.
Dissection and isolation (see Figure 1)
Make sure to minimize the amount of time from the start of dissection to the last step in Dissection and Isolation. Limit the entire Dissection and Isolation within one hour.
Fill one well in the PYREX spot plate with ~1 ml Grace’s media (Figure 1, ‘1. Initial dissection’).
Figure 1. PYREX 9-well spot plate setup
Use forceps to dissect ovary pairs out of eight females in Grace’s media. Make sure not to damage ovaries and keep them intact.
Tip: Gently remove ovaries from the abdomen by grabbing the oviduct/uterus region instead of directly grabbing the ovaries.
Fill another well with ~1 ml Grace’s media and transfer all ovary pairs into this new well (Figure 1, ‘2. Holding ovary pairs’).
See Video 1. ‘Dissection Example’
Move ovary pairs (~two at a time) to a new well with ~1 ml Grace’s media (Figure 1, ‘3. Isolating follicles’).
Use one of the forceps to hold the anterior end of the ovary, near the germarium. Use the other forceps to gently separate ovarioles at the posterior end of the ovary near the calyx.
Next, use the other forcep to gently squeeze follicles out of the posterior end. This process is to liberate follicles from surrounding ovariole sheath. Be careful not to puncture follicles.
Video 1. Dissection example
Identify mature follicles according to the fluorescent reporter and make a pile of intact mature follicles using the needle.
Transfer the intact mature follicles using the P20 pipette to a new well filled with ~1 ml culture media (Figure 1, ‘4. Pool of isolated, intact S14 follicles’). Coat the pipet tip with culture media first to prevent follicles from sticking to the pipet tip.
Repeat Steps C4-C7 until you have enough follicles or until around ~40 min have passed since initial dissection.
Remove any younger-stage follicles in the pool and double check that every follicle in the pool remains intact. Younger-stage follicles should not express the fluorescent reporter when viewed under fluorescent light but can be viewed under the bright-field light.
Use the needle to pile ~25-35 intact follicles together. Transfer piled follicles in culture medium to new wells with a P20 pipette. These follicles should not be dried–they should remain in the ~20 μl culture media in which they were transferred. Repeat this step until all of selected follicles are dispensed in new wells in ‘groups’ of 25-35, or until a total of ~50 min have passed since initial dissection (Figure 1, ‘~25-35 intact S14 follicles in ~20 μl CM’).
Prepare octopamine working solution (see Recipes)–1 ml per each ‘group’.
Add 1 ml of octopamine working solution to each well to stimulate ex vivo rupture OR 1 ml of culture media to each well as a negative control.
Put the PYREX spot plate into a foil-lined utility box (prepared in Procedure B), cover the box, and put the box into a 29 °C incubator for three hours.
Image acquisition
Remove the box from the incubator after three hours.
Use the needle to pile follicles toward the center of each well under a microscope. Do not overlap follicles with each other.
Use Micro-Manager to capture both bright-field and fluorescent images.
Modify for in situ zymography
Follow Steps C1-C10 to prepare isolated mature follicles.
Prepare gelatin working solution (see Recipes)–1 ml per each ‘group’.
Add 1 ml of gelatin working solution to each well to stimulate ex vivo rupture and detect gelatinase activity. A negative control should be gelatin working solution without octopamine.
After three hours incubation at 29 °C, wash out gelatin working solution from each well by pipetting out the solution and replacing with fresh culture media (be careful not to remove follicles from each well).
Use the needle to pile follicles toward the center of each well under the microscope. Do not overlap follicles with each other.
Identify follicles with posterior enzymatic activity (exhibiting green fluorescence at the posterior end) and segregate them to one side. See Video 2 for an example of identifying follicles with posterior enzymatic activity.
Use the micromanager to capture bright-field, green-fluorescent, and red-fluorescent images.
Video 2. Analysis of in situ zymography assay. Open images acquired at the end of each experiment (both bright-field and fluorescent images) in ImageJ. Count the total number of follicles using the multi-point tool in the bright-field image. In this example, there are a total of 29 follicles. Then, count the total number of follicles with posterior green fluorescence in the green-fluorescent image. Before taking the image, most of the follicles identified with enzymatic activity were segregated toward the top of the image. In this example, there are a total of 12 follicles with posterior green fluorescence; therefore, 41.4% of follicles had enzymatic activity.
Data analysis
Data are plotted as ‘Ruptured follicles (%)’. Each data point is one ‘group’ or well: count the total number of follicles in the well and the total number of ruptured follicles (see Video 3, Analysis of ex vivo follicle rupture assay). A follicle is categorized as ‘ruptured’ if > 80% of the oocyte is exposed. Divide the ruptured follicles by the total number of follicles to calculate ‘Ruptured follicles (%)’ (Deady et al., 2015).
For in situ zymography assay, data are plotted as ‘follicles with posterior green fluorescence (%)’. Count the total number of follicles with posterior green fluorescence and divide by the total number of follicles in the well (Deady et al., 2015). (See Video 2, Analysis of in situ zymography assay).
Video 3. Analysis of ex vivo follicle rupture assay. Open images acquired at the end of each experiment (both bright-field and fluorescent images) in ImageJ. Count the total number of follicles using the multi-point tool in the bright-field image. In this example, there are a total of 27 follicles. Then, count the total number of ruptured follicles in the fluorescent image. In this example, there are a total of 20 ruptured follicles; therefore, 74% of follicles ruptured in this example.
Notes
Ruptured follicles (%) from wild-type flies will vary depending upon the fluorescent reporter used. For example, if a reporter only labels the most mature follicles (stage-14C; such as with the R47A04-Gal4 driver; Deady et al., 2017), one would expect to see ~80% of follicles ruptured at the end of the three-hour culture. In contrast, if all stage-14 follicles are labeled and isolated (such as with the R44E10-Gal4 driver; Deady et al., 2017), you would expect to observe ~50% of follicles ruptured. The reduced rupture rate is likely due to the isolation of slightly younger stage-14 follicles that are not competent to OA-induced follicle rupture (Deady et al., 2017).
The ruptured follicles (%) in negative control groups (without octopamine) should be less than 10%. Greater than 10% ruptured follicles in negative controls will indicate that some of the isolated follicles have already initiated the rupture process (such as the breakdown of posterior follicle cells) before adding octopamine working solution. When setting up ex vivo follicle rupture, it is imperative that all follicles are completely intact. For an example of intact, partially ruptured, and ruptured follicles, see Figure 2.
Figure 2. Examples of follicle cell layer coverage of oocyte. Scale bar is estimated 100 μm.
Finish ‘Dissection and Isolation (Procedure C)’ within one hour.
Minimize the amount of endogenous octopamine exposure that follicles receive during isolation. Don’t isolate follicles in the same media that the flies were dissected in; rather, use fresh Grace’s media.
When transferring ~25-35 selected follicles to dry well, make sure they have enough media that they don’t dry out. Usually, the follicles are in ~15-30 μl culture media before adding the octopamine working solution.
Thoroughly mix the octopamine working solution or other drugs before beginning the culture.
During dissections, some follicle-cell layers can envelop the anterior egg chamber in the ovariole (see Figure 2E). Don’t select these egg chambers; they will not rupture.
Recipes
Wet yeast paste
Mix active dry yeast in distilled water. Consistency of the wet yeast paste should be semi-solid, all yeast granules should be dissolved yet yeast should not be watery
Fly food (3 L)
Yeast (g): 61
Cornmeal (g): 163
Molasses (ml): 203
Agar (g): 22
Water (ml): 3,000
5% Tegosept (50 g Tegosept in 1,000 ml ethanol) (ml): 36
Propionic acid (ml): 13
Measure out all ingredients (except Tegosept and Propionic acid), mix thoroughly, and autoclave at 250 °F for 30 min. Once cooled to 70 °C, Tegosept and Propionic acid are added and mixed thoroughly before dispensing to the fly vials
Culture media mixture
Add 1 ml of fetal bovine serum and 100 μl of penicillin-streptomycin (10,000 U/ml) to 9 ml of Grace’s media
Octopamine stock solution (10 mM)
Dissolve octopamine hydrochloride powder in distilled water to 10 mM. Freeze aliquots for up to six months
Octopamine working solution (20 μM)
For each well: 1 ml of culture media mixture + 2 μl of octopamine stock solution. Rock on the nutator for 1~3 min before use
Fluorescein-conjugated DQTM gelatin stock solution (1 mg/ml)
To make aqueous solution, dissolve 1 mg DQTM gelatin powder in 1 ml distilled water and store the solution in fridge covered with foil
Gelatin working solution (25 μg/ml)
For each well: 1 ml of culture media mixture + 25 μl of DG gelatin stock solution + 20 μl of octopamine stock solution. Rock on the nutator for 1~3 min before use
Acknowledgments
We thank current and past members of the Sun lab for suggestions and technical assistance. We are very grateful for Dr. Laurinda Jaffe for initial suggestion of ex vivo culture. JS is supported by the University of Connecticut Start-up fund, NIH/National Institute of Child Health and Human Development Grant R01-HD086175, and Bill and Melinda Gates Foundation. This protocol was implemented in previously published studies (Deady and Sun, 2015; Knapp and Sun, 2017; Deady et al., 2017) and used to identify genes required for ovulation in Drosophila.
The authors declare no conflict of interest.
References
Aigaki, T., Fleischmann, I., Chen, P. S. and Kubli, E. (1991). Ectopic expression of sex peptide alters reproductive behavior of female D. melanogaster. Neuron 7(4): 557-563.
Bloch Qazi, M. C., Heifetz, Y., Wolfner, M. F. (2003). The developments between gametogenesis and fertilization: ovulation and female sperm storage in drosophila melanogaster. Dev Biol 256 195-211.
Cole, S. H., Carney, G. E., McClung, C. A., Willard, S. S., Taylor, B. J. and Hirsh, J. (2005). Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J Biol Chem 280(15): 14948-14955.
Deady, L. D., Shen, W., Mosure, S. A., Spradling, A. C. and Sun, J. (2015). Matrix metalloproteinase 2 is required for ovulation and corpus luteum formation in Drosophila. PLoS Genet 11(2): e1004989.
Deady, L. D. and Sun, J. (2015). A follicle rupture assay reveals an essential role for follicular adrenergic signaling in Drosophila ovulation. PLoS Genet 11(10): e1005604.
Deady, L. D., Li, W. and Sun, J. (2017). The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles. Elife 6: e29887.
Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using muManager software. J Biol Methods 1(2).
Heifetz, Y., Lung, O., Frongillo, E. A., Jr. and Wolfner, M. F. (2000). The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary. Curr Biol 10(2): 99-102.
Knapp, E. and Sun, J. (2017). Steroid signaling in mature follicles is important for Drosophila ovulation. Proc Natl Acad Sci U S A 114(4): 699-704.
Lee, H. G., Rohila, S. and Han, K. A. (2009). The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium. PLoS One 4(3): e4716.
Lee, H. G., Seong, C. S., Kim, Y. C., Davis, R. L. and Han, K. A. (2003). Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster. Dev Biol 264(1): 179-190.
Lim, J, Sabandal, P. R., Fernandez, A., Sabandal, J. M., Lee, H. G., Evans, P. and Han, K. A. (2014). The octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster. PLoS One 9: e104441.
Monastirioti, M. (2003). Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster. Dev Biol 264(1): 38-49.
Monastirioti, M., Linn, C. E., Jr. and White, K. (1996). Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine. J Neurosci 16(12): 3900-3911.
Pfeiffer, B. D., Jenett, A., Hammonds, A. S., Ngo, T.-T. B., Misra, S., Murphy, C., Scully, A., Carlson, J. W., Wan, K. H., Laverty, T. R., et al. (2008). Tools for neuroanatomy and neurogenetics in Drosophila. PNAS 105: 9715-9720.
Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
Spradling, A. C. (1993). Developmental genetics of oogenesis. Cold Spring Harbor Laboratory Press.
Sun, J. and Spradling, A. C. (2013). Ovulation in Drosophila is controlled by secretory cells of the female reproductive tract. Elife 2: e00415.
Copyright: Knapp et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Knapp, E. M., Deady, L. D. and Sun, J. (2018). Ex vivo Follicle Rupture and in situ Zymography in Drosophila. Bio-protocol 8(10): e2846. DOI: 10.21769/BioProtoc.2846.
Deady, L. D., Li, W. and Sun, J. (2017). The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles. Elife 6: e29887.
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Category
Cell Biology > Tissue analysis > Physiology
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2,847 | https://bio-protocol.org/exchange/protocoldetail?id=2847&type=0 | # Bio-Protocol Content
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Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells
SK Sergey Korshunov
JI James A. Imlay
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2847 Views: 6059
Edited by: Valentine V Trotter
Reviewed by: Darrell Cockburn
Original Research Article:
The authors used this protocol in Dec 2015
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Abstract
Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An illustrative example is provided in which Escherichia coli rapidly evolves both cysteine and sulfide upon exposure to exogenous cystine (Chonoles Imlay et al., 2015; Korshunov et al., 2016).
Keywords: Hydrogen sulfide Thiols Cysteine Escherichia coli Measurement Detection
Background
Reduced sulfur species are generated by microbes through several routes. Sulfate-reducing bacteria exploit the reductive process as an integral part of energy generation. Other bacteria release sulfide as a by-product of either the deliberate or adventitious degradation of sulfur species, including cysteine. We have observed that cysteine itself is excreted when intracellular levels are abnormally high, a situation that can occur through uncontrolled amino acid import or dysregulation of cysteine synthesis. These sulfur species are unusually reactive, as they bind metals with high avidity and also are among the few biomolecules that react chemically with molecular oxygen. The upshot is that reduced sulfur compounds can exert important effects upon cells. Hence, it can be important to track the dynamics of reduced sulfur compounds in a variety of contexts.
Thiol agents–notably, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB)–provide good spectroscopic probes of thiol concentrations. Unfortunately, they do not discriminate between organic thiols like cysteine and inorganic species like hydrogen sulfide. The evolution of the latter species has often been detected using lead acetate strips, which are suspended in the head space over sulfide-generating cultures. However, this method is slow and non-quantitative. For that reason, we have leveraged the volatility of hydrogen sulfide so that standard dyes can allow sulfide and organic thiols to be distinguished as they are generated in lab cultures. The methods are simple, quick, and sensitive.
Materials and Reagents
10 ml test tubes, as necessary (Fisher Scientific, catalog number: 14-961-27 )
Polypropylene tubes, 2 ml, as necessary (Denville Scientific, catalog number: C2170 )*
Polypropylene 2 ml tubes, as necessary (see Material and Reagents #2)**
Parafilm* (Bemis, catalog number: PM996 )
Pipette tips (1,000 μl; 200 μl) (Corning, catalog number: 4846 ; USA Scientific, catalog number: 1111-1006 )
Cylinder with compressed air (AirGas Mid-America, breathing quality grade D)
Cylinder with compressed nitrogen (AirGas Mid-America)**
50 ml flasks (Corning, PYREX®, catalog number: 4442-50 )
Flask closures (Fisher Scientific, catalog number: 05-888 )
Bacterial cell culture
Ethylenediamine tetraacetic acid, disodium salt, dihydrate, EDTA (Fisher Scientific, catalog number: S311 )
Cystine dihydrochloride (Sigma-Aldrich, catalog number: C2526 )
5,5-Dithiobis (2-nitrobenzoic acid), DTNB (Sigma-Aldrich, catalog number: D8130 )
4-Amino-N,N-dimethylaniline, DMPDA (Sigma-Aldrich, catalog number: 07750 )
Ferric(III) chloride hexahydrate (Sigma-Aldrich, catalog number: F2877 )
Potassium phosphate, mono- or dibasic (Fisher Scientific, catalog numbers: P284 , P288 )
Ethanol, 100% (Decon Labs, catalog number: 2716 )
Potassium hydroxide (Fisher Scientific, catalog number: P250 )
Glucose (Fisher Scientific, catalog number: D16 )
Ammonium sulfate (Fisher Scientific, catalog number: A702 )
Sodium citrate (Fisher Scientific, catalog number: S279 )
Hydrochloric acid (Sigma-Aldrich, catalog number: H1758 )
Sodium sulfide nonahydrate (Sigma-Aldrich, catalog number: S4766 )
Magnesium sulfate heptahydrate (Fisher Scientific, catalog number: M63 )
Deionized water (University of Illinois deionizing system)
Stock solutions (see Recipes)
Notes:
*These items are for DMPDA-based measurements.
**These items are for DTNB-based measurements.
Equipment
For DMPDA-based measurements
Pipettors, 1 and 0.2 ml (Mettler-Toledo, Rainin, catalog numbers: 17014382 , 17014384 )
Microcentrifuge (Fisher Scientific, model: accuSpinTM Micro 17 , catalog number: 13-100-675)
Spectrophotometer (Beckman Coulter, model: DU-640 )
Shaking water bath (New Brunswick Scientific, model: G76D )
Heater (Fisher Scientific, catalog number: 11-718 )
For DTNB-based measurements
Two 125 ml gas washing bottles with coarse fritted discs (Corning, PYREX®, catalog number: 31760-125C )
Water bath (Shel-Lab, VWR, model: Model 1250 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Korshunov, S. and Imlay, J. A. (2018). Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells. Bio-protocol 8(10): e2847. DOI: 10.21769/BioProtoc.2847.
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Category
Microbiology > Microbial physiology > Stress response
Microbiology > Microbial metabolism > Nutrient transport
Biochemistry > Other compound > Thiol
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2,848 | https://bio-protocol.org/exchange/protocoldetail?id=2848&type=0 | # Bio-Protocol Content
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Hair Follicle Stem Cell Isolation and Expansion
MC Mindy Call
EM Ewa Anna Meyer
WK Winston W. Kao
FK Friedrich E. Kruse
US Ursula Schlӧtzer-Schrehardt
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2848 Views: 7235
Edited by: Vivien Jane Coulson-Thomas
Reviewed by: Anca Savulescu
Original Research Article:
The authors used this protocol in Jan 2011
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Abstract
Stem cells are widely used for numerous clinical applications including limbal stem cell deficiency. Stem cell derived from the bulge region of the hair follicle have the ability to differentiate into a variety of cell types including interfollicular epidermis, hair follicle structures, sebaceous glands and corneal epithelial cells when provided the appropriate cues. Hair follicle stem cells are being studied as a valuable source of autologous stem cells to treat disease. The protocol described below details the isolation and expansion of these cells for eventual clinical application. We used a dual-reporter mouse model to visualize both isolation and eventual differentiation of these cells in a limbal stem cell-deficient mouse model.
Keywords: Holoclones Clonal expansion Hair follicle stem cells Bulge Stem cell isolation
Background
Stem cells are widely used for a multitude of translational and clinical applications. One such clinical application is for the treatment of limbal stem cell deficiency (LSCD). LSCD occurs when there is dysfunction or loss of the limbal stem cell population, which is critical for maintaining a healthy ocular surface, due to congenital or acquired pathologies. The primary treatment strategy for LSCD is cultivating autologous epithelial cell sheets from a limbal biopsy of the patient’s healthy eye (Pellegrini et al., 1997; Shortt et al., 2007). The limitation of this strategy is that it is only applicable for patients that have unilateral LSCD. Those that have bilateral LSCD, must rely on an allogenic limbal biopsy from an immunologically related living donor or cadaveric tissue. Due to the need of systemic immunosuppressive therapy and the limited availability of donor tissue, the therapeutic success rate is decreased. Several research groups have been examining the use of cultivated oral mucosal cells for the treatment of LSCD and have achieved some success. However, these cells often fail to express the corneal epithelial differentiation marker, Keratin 12 (Inatomi et al., 2006) and often result in the development of peripheral neovascularization (Nakamura et al., 2004; Nishida et al., 2004; Ma et al., 2009). Due to these limitations, there was a need for an alternative source of autologous stem cells. Thus we focused on the use of hair follicle stem cells as they harbor multiple sources of stem cells that have been used in regenerative medicine (Cotsarelis et al., 1990; Purba et al., 2014). The hair follicle contains mesenchymal stem cells in the dermal papilla and connective tissue sheath, which can give rise to several cell lineages (Lako et al., 2002; Jahoda et al., 2003; Richardson et al., 2005). Additionally, the bulge region of the hair follicle contains stem cells, which can generate the interfollicular epidermis, hair follicle structures and sebaceous glands (Cotsarelis et al., 1990; Taylor et al., 2000; Cotsarelis, 2006). The hair follicle stem cells (HFSC) derived from the bulge region express of variety of cytokeratins including cytokeratin 15 (Krt15) (Tiede et al., 2007; Kloepper et al., 2008; Larouche et al., 2008), which has been successfully used for the purification and enrichment of HFSC (Blazejewska et al., 2009). HFSC have been successfully used in the treatment of a mouse model of LSCD (Meyer-Blazejewska et al., 2011) and research continues to focus on other therapeutic applications and the eventual translation to humans (Purba et al., 2014). Continued research efforts into these areas rely on a standard method for isolating and expanding the bugle-derived HFSC.
Materials and Reagents
Pipette tips (MidSci, Avant low binding tips)
35-mm cell culture dish (Thermo Fisher Scientific, catalog number: 153066 )
6-well plates (Corning, Falcon®, catalog number: 353934 )
100 mm cell culture dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150464 )
NIH-3T3 cells (ATCC, catalog number: CRL-1658 )
3-5 week old K12rtTA/rtTA/TetO-Cre/RosamTmG (see Notes)
Ketamine/HCl 100 mg/ml (KetaJect; Henry Schein Animal Health, catalog number: 010177 )
Xylazine AnaSed® 100 mg/ml (Santa Cruz Biotechnology, catalog number: sc-362949Rx )
Collagenase A (Sigma-Aldrich, Roche Diagnostics, catalog number: 10103578001 )
Dispase II (Sigma-Aldrich, catalog number: 4942078001)
Manufacturer: Roche Diagnostics, catalog number: 04942078001 .
Mitomycin C (Sigma-Aldrich, catalog number: M7949-2MG )
Phosphate buffered saline (PBS)
Trypsin (2.5%) (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
Versene (Thermo Fisher Scientific, GibcoTM, catalog number: 15040066 )
Dulbecco’s modified Eagle medium (DMEM) without calcium and magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 21068028 )
Ham’s F12 Nutrient Mix (Thermo Fisher Scientific, GibcoTM, catalog number: 11765047 )
Fetal Bovine Serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
Human recombinant epidermal growth factor (Merck, catalog number: GF144 )
L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
Calcium Chloride solution 1 M (Sigma-Aldrich, catalog number: 21115 )
Human corneal growth supplement (Thermo Fisher Scientific, GibcoTM, catalog number: S0095 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
Amphotericin B (Thermo Fisher Scientific, catalog number: 15290026 )
Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
Stem Cell Media (see Recipes)
3T3 media (see Recipes)
Equipment
Pipettes
Microdissection scissors (Fine Science Tools, catalog number: 15000-00 )
Forceps (Fine Science Tools, catalog number: 11252-23 )
Scissors (Fine Science Tools, catalog number: 14060-09 )
Hemocytometer (Hausser Scientific, catalog number: 3200 )
Dissecting Scope (ZEISS, model: Stemi DV4 )
BSL2 Laminar flow hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series A2 , catalog number: 1387)
CO2 incubator (Thermo Fisher Scientific, Thermo ScienticTM, model: NAPCO Series 8000 WJ )
Centrifuge (Hettich, model: Rotina 35 )
Inverted fluorescent microscope (Zeiss Observer Z1 with an apotome attachment) (ZEISS, model: AxioObserver Z1 )
Software
AxioVison 4.7
ImageJ
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Call, M., Meyer, E. A., Kao, W. W., Kruse, F. E. and Schloetzer-Schredhardt, U. (2018). Hair Follicle Stem Cell Isolation and Expansion. Bio-protocol 8(10): e2848. DOI: 10.21769/BioProtoc.2848.
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Category
Stem Cell > Adult stem cell > Epithelial stem cell
Cell Biology > Cell isolation and culture > Cell differentiation
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2,849 | https://bio-protocol.org/exchange/protocoldetail?id=2849&type=0 | # Bio-Protocol Content
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Murine Hair Follicle Derived Stem Cell Transplantation onto the Cornea Using a Fibrin Carrier
MC Mindy Call
EM Ewa Anna Meyer
WK Winston W. Kao
FK Friedrich E. Kruse
US Ursula Schlӧtzer-Schrehardt
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2849 Views: 5430
Edited by: Vivien Jane Coulson-Thomas
Reviewed by: Dongsheng Jiang
Original Research Article:
The authors used this protocol in Jan 2011
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Abstract
The goal of this protocol is to establish a procedure for cultivating stem cells on a fibrin carrier to allow for eventual transplantation to the eye. The ability to transfer stem cells to a patient is critical for treatment for a variety of disorders and wound repair. We took hair follicle stem cells from the vibrissae of transgenic mice expressing a dual reporter gene under the control of the Tet-on system and the keratin 12 promoter (Meyer-Blazejewska et al., 2011). A clonal growth assay was performed to enrich for stem cells. Once holoclones formed they were transferred onto a fibrin carrier and cultivated to obtain a confluent epithelial cell layer. Limbal stem cell deficient (LSCD) mice were used as the transplant recipient in order to test for successful grafting and eventual differentiation into a corneal epithelial phenotype.
Keywords: Holoclones Clonal growth Hair follicle stem cells Transgenic mice Fibrin carrier
Background
Stem cells are widely used as a therapeutic tool, thus a means for delivery is essential. In fact, many researchers and companies are searching for the best way to deliver cells into the human body to optimize cell survival as well as integration into the host tissue. Injection methods have been widely used in animal models but often result in poor survival and integration. Techniques utilizing biomaterials and surgical devices are currently being employed. One technique that has been utilized to deliver stem cells is fibrin carriers. Fibrin gel is a degradable biopolymer that can adhere to native tissue allowing for cell attachment, migration and proliferation (Ehrbar et al., 2005). Fibrin gels have many advantages including biocompatibility, controlled degradation (Kjaergard et al., 1994; Sidelmann et al., 2000), uniform cell distribution and high cell seeding efficiency (Swartz et al., 2005). Fibrin gels have been utilized for treating skin burns (Pellegrini et al., 1999; Ronfard et al., 2000), junctional epidermolysis bullosa (Hirsch et al., 2017) and corneal damage (Pellegrini et al., 1997; Rama et al., 2010). The method described here uses a fibrin carrier to transplant hair follicle derived stem cells onto the ocular surface of a limbal stem cell-deficient mouse. Cell engraftment and differentiation was assessed for a 5-week period via fluorescent microscopy.
Materials and Reagents
TISSEEL [Fibrin Sealant] (Baxter, catalog number: 1501261 )
Insulin syringe (Fisher Scientific, catalog number: 14-829-1A)
Manufacturer: BD, catalog number: 329420 .
6-well plates (Corning, Falcon®, catalog number: 353934 )
Pipette tips (MidSci, Avant low binding tips)
Transfer pipet
HFSC discs
Ethilon 10-0 nylon sutures (Ethicon, catalog number: 9006G )
Microscope slides (Fisher Scientific, catalog number: 12-552-3 )
Coverglass 22 x 50 (Fisher Scientific, catalog number: 12-548-5E )
Thrombin
C57BL/6 mice (THE JACKSON LABORATORY, catalog number: 000664 )
K12rtTA/rtTA/tetO-cre/ROSAmTmG transgenic mice (see Notes)
0.9% saline (Fisher Scientific, catalog number: 23-535435 )
BioGlo fluorescein sodium ophthalmic strips (Hub Pharmaceuticals, NDC 17238-900-11)
Doxycycline chow (Custom Animal Diets, catalog number: AD3008 )
Avastin (bevacizumab, Genentech, Inc.)
Anti-inflammatory drops–Inflanefran forte (Allergan, NDC 11980-180)
16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
Sodium borohydride (Sigma-Aldrich, catalog number: 71320 )
DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3571 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-1 )
Calcium chloride (CaCl2) (Acros Organics, catalog number: 349610025 )
Dulbecco’s modified Eagle medium (DMEM) without calcium and magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 21068028 )
Ham’s F12 Nutrient Mix (Thermo Fisher Scientific, GibcoTM, catalog number: 11765047 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
Human recombinant epidermal growth factor (Merck, catalog number: GF144 )
L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
Human corneal growth supplement (Thermo Fisher Scientific, GibcoTM, catalog number: S0095 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
GibcoTM Amphotericin B (Thermo Fisher Scientific, catalog number: 15290026 )
Sodium phosphate dibasic, anhydrous (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
Sodium phosphate monobasic, anhydrous (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
Analytical grade glycerol (Sigma-Aldrich, catalog number: G6279 )
Mowiol 4-88 (Merck, catalog number: 475904 )
Tris (Fisher Scientific, catalog number: BP152-1 )
Ketamine/HCl 100 mg/ml (KetaJect; Henry Schein Animal Health, catalog number: 010177 )
Xylazine AnaSed® 100 mg/ml (Santa Cruz Biotechnology, catalog number: sc-362949Rx )
Fibrinogen solution (see Recipes)
Thrombin solution (see Recipes)
Stem cell media (see Recipes)
0.1 M Phosphate Buffer, pH 7.4 (see Recipes)
Mowiol mounting medium (see Recipes)
Ketamine/xylazine solution (see Recipes)
Equipment
Algerbrush II Corneal rust ring remover (MicroSurgical Technology, catalog number: AM0100 )
Inverted Fluorescence Microscope (Zeiss Observer Z1 with an apotome attachment) (ZEISS, model: AxioObserver Z1 )
Suture Tying Forceps (Fine Science Tools, catalog number: 18025-10 )
Microdissection scissors (Fine Science Tools, catalog number: 15000-00 )
Epi-fluorescent stereomicroscope (ZEISS, model: Stemi SVII )
BSL2 Laminar Flow Hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series A2 , catalog number: 1387)
Hemocytometer (Hausser Scientific, catalog number: 3200 )
CO2 Incubator (Thermo Fisher Scientific, Thermo ScienticTM, model: NAPCO Series 8000 WJ )
Dissecting Scope (ZEISS, model: Stemi DV4 )
Centrifuge (Hettich, model: Rotina 35 )
Software
AxioVison 4.7
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Call, M., Meyer, E. A., Kao, W. W., Kruse, F. E. and Schloetzer-Schredhardt, U. (2018). Murine Hair Follicle Derived Stem Cell Transplantation onto the Cornea Using a Fibrin Carrier. Bio-protocol 8(10): e2849. DOI: 10.21769/BioProtoc.2849.
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Category
Stem Cell > Adult stem cell > Epithelial stem cell
Stem Cell > Adult stem cell > Cell transplantation
Cell Biology > Cell Transplantation > Allogenic Transplantation
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285 | https://bio-protocol.org/exchange/protocoldetail?id=285&type=0 | # Bio-Protocol Content
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Peer-reviewed
Establishing Primary Malignant Pleural Mesothelioma (MPM) Cell Cultures
MC Mario Cioce
CC Claudia Canino
SS Sabrina Strano
GB Giovanni Blandino
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.285 Views: 14753
Original Research Article:
The authors used this protocol in Jun 2012
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Jun 2012
Abstract
This is a general protocol for the isolation and maintenance of primary MPM cultures as a tool for the identification of Tumor Initiating Cells and early progenitor-targeting drugs (Cioce et al., 2010). Primary cultures can be propagated efficiently for 8-12 weeks and xenotransplanted in NOD/SCID mice while retaining the histofeatures of the originating tumor (Canino et al., 2012). The protocol is suitable for both MPM solid specimens and pleural effusion. For increased clarity, initially two separate sections addressing the isolation of MPM cells from solid tumors and pleural effusions are here provided.
Materials and Reagents
Surgical specimen or pleural effusion
Phosphate buffered saline (PBS)
Collagenase type XI (300 U/ml) (Sigma-Aldrich, catalog number: C9407 )
Hyaluronidase (100 U/ml) (Sigma-Aldrich, catalog number: H4272 )
DMEM/F12 (1:1)+ GLUTAMAX (Life Technologies, Invitrogen™, catalog number: 10565-018 )
FBS-non heat inactivated (Life Technologies, catalog number: 10437-028 )
Human recombinant insulin (Sigma-Aldrich, catalog number: I-5500 )
Bovine Serum Albumin-Fatty Acid Free (BSA-FAF) (Sigma-Aldrich, catalog number: A7030 )
Non-heat inactivated FBS (Life Technologies, Gibco®, catalog number: 16000-044 )
Ciprofloxacin (Sigma-Aldrich, catalog number: 17850 )
Red blood lysis buffer (0.8% ammonium chloride) (Life Technologies, catalog number: A10492-01 )
ACCUTASE (STEMCELL Technologies, catalog number: AT-104 )
0.4% trypan blu (Life Technologies, Gibco®, catalog number: 15250-061 )
Digestion medium
Equipment
Cell culture set up
Scalpels (Becton, Dickinson and Company, catalog number: 371621 )
Microdissecting forceps
5 ml Pasteur pipette (BD Biosciences, Falcon®)
15 ml centrifuge tubes
50 ml centrifuge tubes
Centrifuge capable of running at ≥ 300 x g
70 μm nylon mesh (BD Biosciences, Falcon®, catalog number: 352350 )
Ultralow attachment dishes (Corning Incorporated, catalog number: 3261 for 100 mm) (or alternatively, sterile Petri dishes non treated for cell culture)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Cioce, M., Canino, C., Strano, S. and Blandino, G. (2012). Establishing Primary Malignant Pleural Mesothelioma (MPM) Cell Cultures. Bio-protocol 2(21): e285. DOI: 10.21769/BioProtoc.285.
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Category
Cancer Biology > General technique > Cell biology assays
Cell Biology > Cell isolation and culture > Cell differentiation
Cell Biology > Cell isolation and culture > Cell isolation
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2,850 | https://bio-protocol.org/exchange/protocoldetail?id=2850&type=0 | # Bio-Protocol Content
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Peer-reviewed
Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism
BP Birte Plitzko
Sandra Loesgen
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2850 Views: 72265
Reviewed by: Amriti Rajender Lulla
Original Research Article:
The authors used this protocol in Oct 2017
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Oct 2017
Abstract
Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs (Koppenol et al., 2011; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza et al., 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in living cells, utilizing the Seahorse XF24 Extracellular Flux Analyzer (Figure 1). The Seahorse XF24 Extracellular Flux Analyzer continuously measures oxygen concentration and proton flux in the cell supernatant over time (Wu et al., 2007). These measurements are converted in OCR and ECAR values and enable a direct quantification of mitochondrial respiration and glycolysis. With this protocol, we sought to assess basal mitochondrial function and mitochondrial stress of three different cancer cell lines in response to the cytotoxic test lead compound mensacarcin in order to investigate its mechanism of action. Cells were plated in XF24 cell culture plates and maintained for 24 h. Prior to analysis, the culture media was replaced with unbuffered DMEM pH 7.4 and cells were then allowed to equilibrate in a non-CO2 incubator immediately before metabolic flux analysis using the Seahorse XF to allow for precise measurements of Milli-pH unit changes. OCR and ECAR were measured under basal conditions and after injection of compounds through drug injection ports. With the described protocol we assess the basic energy metabolism profiles of the three cell lines as well as key parameters of mitochondrial function in response to our test compound and by sequential addition of mitochondria perturbing agents oligomycin, FCCP and rotenone/antimycin A.
Figure 1. Overview of seahorse experiment
Keywords: Bioenergetics Seahorse XF Mitochondrial metabolism Glycolysis Mitochondrial respiration
Background
Natural products are small molecules that are isolated from natural sources. Over the last century, these molecules have been instrumental in treating human diseases, especially inspired chemotherapeutics. Metabolites like taxol, vincristine, and doxorubicin have revolutionized how we treat malign cancers and other natural products, for example rapamycin, oligomycin, and bafilomycin, are used as molecular probes and enable molecular studies of biochemical and cellular processes in the laboratory. While studying the mechanism of action of the cytotoxic natural product mensacarcin, we found that a fluorescently labeled mensacarcin probe localizes to a great extent in mitochondria (Plitzko et al., 2017). To investigate if mensacarcin’s cytotoxic properties might be derived from interference with mitochondrial function, we sought to examine mensacarcin’s effects on cellular bioenergetics. Using a Seahorse Extracellular Flux Analyzer, we monitored cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) in real time as measures of mitochondrial respiration and glycolysis, respectively (Wu et al., 2007; Serill et al., 2015). The Seahorse XF24 Extracellular Flux Analyzer allows continuous direct quantification of mitochondrial respiration and glycolysis of living cells. The instrument uses a sensor cartridge in a 24-well plate format with each sensor being equipped with two embedded fluorophores: one which is quenched by oxygen (O2) and the other that is sensitive to change in pH. During measurements, the sensor cartridge is lowered 200 µm above the cell monolayer, forming a micro-chamber of about 2 µl. The Seahorse instrument contains fiber optic bundles that emit light, excite the fluorophores, and then measures the change in the fluorophore’s emission. The very small test volume formed by the transient micro chamber allows for sensitive, precise, and nondestructive measurements of parameters in real time. Changes in oxygen concentration and pH are automatically calculated and reported as Oxygen Consumption Rate (OCR) and Extra Cellular Acidification Rate (ECAR). Once a measurement is completed, the sensors lift which allows the larger medium volume above to mix with the medium in the transient micro chamber, restoring values to baseline. The sensor cartridge contains ports that allow sequential addition of up to four compounds per well during the assay measurements.
With the described protocol we assessed the energy metabolism of three cell lines (HCT-116, SK-Mel-28, and SK-Mel-5) (Figure 6). Addition of mensacarcin was found to have pronounced effect on the basal OCR of melanoma cells and no increasing effect on ECAR. An increase in glycolysis is often observed as a compensatory response. Mitochondria are essential for the energy metabolism of cells and have a key role in apoptotic cell death. Alteration of the mitochondrial respiration or the equilibrium between the pro-apoptotic and anti-apoptotic proteins can induce mitochondrial failure. To gain insights into the induced mitochondrial impairment in melanoma cells, we assessed key parameters of mitochondrial respiration by consecutively exposing cells to well described mitochondria perturbing reagents. Following addition of our test compound mensacarcin, we sequentially added oligomycin, FCCP, and lastly rotenone and antimycin A (Figure 5). Oligomycin inhibits ATP synthase and reduces OCR, FCCP uncouples oxygen consumption from ATP production and raises OCR to a maximal value, and antimycin A and rotenone target the electron transport chain and reduce OCR to a minimal value. The mitochondria stress test protocol provides information on basal respiration, ATP-linked respiration, proton leak, maximal respiration capacity, and non-mitochondrial respiration of cells. Therefore, this assay can be used to provide insight on the mechanism of action of compounds that directly target mitochondrial respiration.
Traditional measurements of mitochondrial function or glycolysis require an oxygen electrode, or kits and dyes that utilize colorimetric or fluorimetric detection (Li and Graham, 2012; TeSlaa and Teitell, 2014). Most of these methods are invasive and cumbersome methods that only allow low sample throughput. In contrast, the Seahorse analyzer assay with its sensor cartridge system enables measurement of mitochondrial respiration and glycolysis in real time and in a non-invasive manner that does not require any dyes or labels. Cellular energy metabolism research is highly topical in all fields of mammalian cell biology. The following protocol was developed for researchers in cancer biology but with approaches that suit studies of energy metabolism in all mammalian cell systems.
Materials and Reagents
CELLSTAR® Tissue Culture Plates, 96-well (Greiner Bio One International, catalog number: 655180 )
Sterile racked pipette tips (1 ml and 200 μl) (VWR, catalog numbers: 613-0738 ; 613-0742 )
Sterile basins (Corning, Costar®, catalog number: 4870 )
Sterile reagent tubes (15 and 50 ml) (VWR, catalog numbers: 89039-668 ; 89039-662 )
Sterile Serological pipettes (5, 10, 25, 50 ml) (Fisher Scientific, catalog numbers: 13-678-11 , 13-678-11D , 13-678-11E , 13-678-11F )
Glass bottles (500 ml) (Fisher Scientific, catalog number: FB8001000 )
HCT-116, SK-Mel-5 and SK-Mel-28 cells (ATCC, catalog numbers: CCL-247 , HTB-70 , HTB-72 )
Seahorse XF24 FluxPak (including sensor cartridges, tissue culture plates, calibrant solution and calibration plates) (Agilent Technologies, Santa Clara, CA)
Trypsin/EDTA (0.25%/2.21 mM) (Corning, catalog number: 25-053-Cl )
1x Ca2+/Mg2+-free DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
Liquid Dulbecco’s modified Eagle’s medium (DMEM) (Corning, catalog number: 10-013 )
Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
Penicillin/streptomycin solution 100x (Corning, catalog number: 30-002-Cl )
Powder Dulbecco’s modified Eagle’s medium (DMEM) without Na2HCO3, without HEPES (Corning, catalog number: 50-013 )
Sodium hydroxide (NaOH) (VWR, catalog number: 97064-476 )
Oligomycin (Merck, catalog number: 495455-10MG )
DMSO (VWR, catalog number: BDH1115-1LP )
FCCP (Cayman Chemical, catalog number: 15218 )
Rotenone (Cayman Chemical, catalog number: 13995 )
Antimycin A (Enzo Life Sciences, catalog number: ALX-380-075-M005 )
Culture media (10% (v/v) FBS) (see Recipes)
Assay media (see Recipes)
NaOH (1 M) (see Recipes)
Oligomycin (10 µM) (see Recipes)
FCCP (5 µM) (see Recipes)
Rotenone (5 µM)/antimycin A (5 µM) (see Recipes)
Equipment
Hemacytometer (Hausser Scientific, catalog number: 1490 )
Biological Safety Cabinet Class II, Type A2 (NuAire, model: NU-425-400ES )
Seahorse XF Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA)
Pipet-Lite Pipette XLS STD 20 XLS (Mettler Toledo, Rainin, model: SL-2XLS+ )
Pipet-Lite Pipette XLS STD 200 (Mettler Toledo, Rainin, model: SL-200XLS+ )
Pipet-Lite Pipette XLS 1000 (Mettler Toledo, Rainin, model: SL-1000XLS+ )
Multichannel Pipet-Lite Pipette XLS 8-CH 1200 (Mettler Toledo, Rainin, model: L8-1200XLS+ )
Multichannel Pipet-Lite Pipette XLS 8-CH 200 (Mettler Toledo, Rainin, model: L8-200XLS+ )
Aspirator pump
Humidified non-CO2 incubator (XF Prep Station; Agilent Technologies, Santa Clara, CA)
Shallow water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: Precision 180 )
Pipette controller (BrandTech Scientific, model: Accu-Jet® Pro , catalog number: 26330)
Humidified, 37 °C, 5% CO2 incubator (Eppendorf, model: Galaxy® 170 R )
-20 °C biomedical freezer (Sanyo, model: MDF-U731M )
Autoclave (Consolidated Sterilizer Systems, model: SSR-3A , ADVPB)
Inverted light microscope (Nikon Instruments, model: Eclipse TS100 )
pH-meter with semi-micro electrode (Thermo Fisher Scientific, Thermo ScientificTM, model: Orion StarTM A211 , with ROSS 8103BN electrode: (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 8103BN )
Software
Seahorse Bioscience XF24 software
Excel (Microsoft)
GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Plitzko, B. and Loesgen, S. (2018). Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism. Bio-protocol 8(10): e2850. DOI: 10.21769/BioProtoc.2850.
Plitzko, B., Kaweesa, E. N. and Loesgen, S. (2017). The natural product mensacarcin induces mitochondrial toxicity and apoptosis in melanoma cells. J Biol Chem 292(51): 21102-21116.
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Category
Cancer Biology > Cellular energetics > Cell biology assays
Cell Biology > Cell metabolism > Other compound
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2,851 | https://bio-protocol.org/exchange/protocoldetail?id=2851&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
High Dimensional Functionomic Analysis of Human Hematopoietic Stem and Progenitor Cells at a Single Cell Level
TL Thomas Luh
KL Kimberly Lucero
WM Wenji Ma
JL Jaeyop Lee
YZ Yu Jerry Zhou
YS Yufeng Shen
Kang Liu
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2851 Views: 7181
Edited by: Ruth A. Franklin
Reviewed by: Lokesh KalekarChris Tibbitt
Original Research Article:
The authors used this protocol in Jul 2017
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Abstract
The ability to conduct investigation of cellular transcription, signaling, and function at the single-cell level has opened opportunities to examine heterogeneous populations at unprecedented resolutions. Although methods have been developed to evaluate high-dimensional transcriptomic and proteomic data (relating to cellular mRNA and protein), there has not been a method to evaluate corresponding high-dimensional functionomic data (relating to cellular functions) from single cells. Here, we present a protocol to quantitatively measure the differentiation potentials of single human hematopoietic stem and progenitor cells, and then cluster the cells according to these measurements. High dimensional functionomic analysis of cell potential allows cell function to be linked to molecular mechanisms within the same progenitor population.
Keywords: CD34+ hematopoietic stem and progenitor cells Single cell culture Differentiation Quantitative clonal output Barnes-Hut t-SNE Dimension reduction
Background
The development of techniques for single-cell measurements of cell transcription, signaling, and function at the single-cell level, alongside preexisting technologies such as flow cytometry, has allowed new lenses to examine complex, heterogeneous populations. Such methods generate large amounts of data, which can be interpreted with aid from dimensionality reduction algorithms, as illustrated on single-cell RNA-Seq using Mpath, Monocole, PCA, Wishbone, or diffusion map algorithms (Paul et al., 2016; See et al., 2017), and on CyTOF using tSNE or PhenoGraph (Amir el et al., 2013; Levine et al., 2015).
We developed this protocol to allow functional analysis and subsequent dimension reduction of large-scale culture of hematopoietic progenitors in single-cell environments. In this protocol, we describe a method to culture single cells of human CD34+ hematopoietic stem and progenitor cells (HSPCs) in a stromal cell culture with cytokines, to enumerate the clonal outcome (functionomics) of six different lineages (i.e., granulocyte, monocyte, lymphocyte, CD141+ dendritic cell (conventional type 1 DC (cDC1)), CD1c+ dendritic cell (conventional type 2 DC (cDC2)), and plasmacytoid dendritic cells (pDC)) from each progenitor, and to cluster the progenitors according to function with a dimension reduction method. In our previous paper, we showed that this is feasible for a population of 2,247 progenitors; each progenitor can be individually plotted to form a two-dimensional ‘map’ of 2,247 data points (Lee et al., 2017). Our protocol allows for the functional clustering of single cells. Such high dimensional functionomic analysis aids in linking cell function to molecular mechanism of any given cell population.
Materials and Reagents
15 cm culture plates (Corning, Falcon®, catalog number: 353025 )
15 ml Falcon tubes (Corning, Falcon®, catalog number: 352097 )
96-well V-bottom plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 249570 )
Flat-bottom 96-well plates (Corning, Falcon®, catalog number: 353072 )
50 ml Falcon tubes (Corning, Falcon®, catalog number: 352098 )
Serological pipettes (Fisher Scientific, catalog number: 13-678-11 )
Opaque Eppendorf tubes (CELLTREAT Scientific Products, catalog number: 229437 )
Eppendorf tubes (Fisher Scientific, catalog number: 05-408-129 )
Pipette tips
Pasteur pipette (Fisher Scientific, catalog number: 13-678-8B )
100 μm cell strainer (Corning, Falcon®, catalog number: 352360 )
0.20 μm filter (Corning, catalog number: 431229 )
MS5 (Lee et al., 2015a and 2015b)
OP9 (Lee et al., 2015a and 2017)
dH2O (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977023 )
Trypsin (Corning, catalog number: 25-052-CI )
Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
70% ethanol
Ficoll-Paque Plus (GE Healthcare, catalog number: 17144002 )
Anti-human CD34 MACS microbeads (Miltenyi Biotec, catalog number: 130-046-702 )
Recombinant human Flt3L (Celldex Therapeutics, catalog number: CDX-301 )
Recombinant human SCF (PeproTech, catalog number: 300-07 )
Recombinant human GM-CSF (PeproTech, catalog number: 300-03 )
Antibodies (details of clone name, fluorochrome, manufacturer and dilution are listed in Tables 1 and 3)
MEMα with deoxyribonucleosides (Thermo Fisher Scientific, GibcoTM, catalog number: 12571071 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
Pen-Strep (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Mitomycin C (Sigma-Aldrich, catalog number: M4287-5X2MG )
Dulbecco’s phosphate-buffered saline (DPBS) (GE Healthcare, catalog number: SH30378.02 )
Ethylenediaminetetraacetic acid (EDTA) (Corning, catalog number: 46-034-CI )
Bovine serum albumin (BSA) (ThermoFisher, catalog number: BP1600-100 )
FcR blocking buffer (Miltenyi Biotec, catalog number: 130-059-901 )
CountBright Absolute Counting Beads (Thermo Fisher Scientific, InvitrogenTM, catalog number: C36950 )
Complete MEMα medium (see Recipes)
Mitomycin C stock (1 mg/ml) (see Recipes)
FACS buffer (see Recipes)
Anti-CD34 microbeads/FcR blocking mix (see Recipes)
Equipment
Pipettes
Incubator (Forma Scientific, model: 3354 )
Centrifuge (Eppendorf, model: 5810 R , catalog number: 5811000320)
Vortex with universal holder (VWR, catalog number: 97043-562 )
Microscope
Flow cytometer (BD, models: BD LSRII or LSRFortessaTM )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Luh, T., Lucero, K., Ma, W., Lee, J., Zhou, Y. J., Shen, Y. and Liu, K. (2018). High Dimensional Functionomic Analysis of Human Hematopoietic Stem and Progenitor Cells at a Single Cell Level. Bio-protocol 8(10): e2851. DOI: 10.21769/BioProtoc.2851.
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Category
Immunology > Immune cell differentiation > HSPCs
Stem Cell > Adult stem cell > Hematopoietic stem cell
Cell Biology > Single cell analysis > Flow cytometry
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2,852 | https://bio-protocol.org/exchange/protocoldetail?id=2852&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development
CY Changfu Yao*
GC Gianni Carraro*
BS Barry R. Stripp
*Contributed equally to this work
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2852 Views: 6241
Edited by: Giusy Tornillo
Reviewed by: Zhenguang ZhangGunjan Mehta
Original Research Article:
The authors used this protocol in Jan 2017
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Jan 2017
Abstract
Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide up-to-date protocols for the establishment of this culture model and its application to investigate hedgehog signaling in the developing lung.
Keywords: Mouse embryonic lung In vitro Explant culture
Background
Mouse lung development initiates as an endodermal diverticulum of the anterior foregut endoderm at day 9.5 postcoitum (E9.5), with subsequent closure of a proximal tracheoesophageal septum for the formation of distinct tracheal and esophageal tubes (Minoo and King, 1994). Subsequent branching of primitive endodermal tubes yields a planar lung structure by E12.5, with subsequent orthogonal branches yielding three-dimensional structure characteristic of the mature lung (Metzger et al., 2008). The planar structure of lung rudiments isolated prior to E12.5 is suitable for in vitro culture at an air liquid interface (Carraro et al., 2010; Del Moral and Warburton, 2010). Embryonic lung is isolated by dissection using a stereo microscope either under bright field illumination or by fluorescence illumination when coupled with lineage tracing and fluorescent reporters. Herein we describe the use of a ShhCre/RosamTmG reporter mouse allowing Cre-mediated activation of membrane-localized GFP within anterior foregut endoderm from approximately E8.75 (Montgomery et al., 2007; Goss et al., 2009; Yao et al., 2017). Accordingly lung endoderm is visualized by green fluorescence and surrounding tissue by red fluorescence, allowing clear identification and microdissection of developing endodermal structures, including the lung, and imaging during in vitro culture.
Materials and Reagents
Whole embryonic lung isolation
BD 1 ml TB syringe 26 G (BD, catalog number: 309625 )
50 ml conical tube (Denville Scientific, catalog number: C1062-P (1000799))
Petri dish (Greiner Bio One International, catalog number: 663161 )
ShhCre mice (THE JACKSON LABORATORY, catalog number: 005622 )
RosamTmG/+ mice (THE JACKSON LABORATORY, catalog number: 007576 )
Mouse embryonic lungs from ShhCre/+, RosamTmG/+ mice
Note: Mouse embryonic lungs from ShhCre/+; RosamTmG/+ mice were harvested between E10.5 and E12.5. The day of vaginal plug detection was considered to be E0.5
General anesthesia: Ketamine (VET one, NDC 13985-702-10) and xylazine (AnaSed Injection, NDC 59339-110-20)
70% ethanol (Fisher Scientific, catalog number: BP8201500 )
Phosphate buffered saline (PBS) (1x), liquid, without calcium and magnesium (Corning, catalog number: 21-040-CV )
Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
PBS with P/S (see Recipes)
Whole embryonic lung culture
12 wells plate (Denville Scientific, catalog number: T1012 )
Disposable transfer pipettes, sterile (VWR, catalog number: 414004-036 )
Razor blade (VWR, catalog number: 55411-050 )
Nuclepore Polycarbonate Track-Etch membrane (13 mm, 8 μm) (GE Healthcare, catalog number: 150446 )
Dulbecco’s modified Eagle medium: Nutrient Mix F-12 (DMEM/F12) (1x), liquid, 1:1 Contains GlutaMAX, but no HEPES buffer (Thermo Fisher Scientific, GibcoTM, catalog number: 10565042 )
Penicillin-streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
BenchMark fetal bovine serum (Gemini Bio-Products, catalog number: 100-106 )
Embryonic lung culture medium (see Recipes)
Equipment
Surgical instruments including:
2 Dumont #5 forceps (Fine Science Tools, catalog number: 11295-10 )
Moria® spoon (perforated) (Fine Science Tools, catalog number: 10370-17 )
Mouse necropsy instrument set including:
Metzenbaum (Lahey) Scissors (Roboz Surgical Instrument, catalog number: RS-6950 )
Micro Dissecting Scissors 4" Blunt (Roboz Surgical Instrument, catalog number: RS-5980 )
Graefe Forceps Straight (Roboz Surgical Instrument, catalog number: RS-5130 )
Graefe Forceps Curved (Roboz Surgical Instrument, catalog number: RS- 5135 )
CO2 Incubator (Thermo Fisher Scientific, model: HeracellTM 150i )
Fluorescent Stereo Microscope (Carl Zeiss, model: Zeiss SteREO Discovery.V8 , with 8x magnification) equipped with 5 MP, 36 bit, Peltier cooled Zeiss Axiocam MRc5 camera (Carl Zeiss, model: AxioCam MRc 5 )
Software
Zen blue software (Carl Zeiss)
PRISM software version 7
Procedure
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How to cite：Yao, C., Carraro, G. and Stripp, B. R. (2018). In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development. Bio-protocol 8(10): e2852. DOI: 10.21769/BioProtoc.2852.
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Category
Developmental Biology > Morphogenesis > Organogenesis
Cell Biology > Tissue analysis > Tissue isolation
Cell Biology > Cell imaging > Fluorescence
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2,853 | https://bio-protocol.org/exchange/protocoldetail?id=2853&type=0 | # Bio-Protocol Content
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Peer-reviewed
Sociability and Social Novelty Preference Tests Using a U-shaped Two-choice Field
EL Eun-Hwa Lee
JP Jin-Young Park
YL Yunjin Lee
PH Pyung-Lim Han
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2853 Views: 10377
Edited by: Oneil G. Bhalala
Reviewed by: Arnau Busquets-Garcia
Original Research Article:
The authors used this protocol in Sep 2017
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Abstract
We developed sociability tests based on use of a U-shaped two-choice field to read out behavioral states of sociability in rodents. The U-shaped two-choice field is a modified open field that is partially partitioned with a wall projecting to the central point, resulting in two symmetrical rectangular fields, each containing closed and open square zones that together form a ‘U-shaped field’. The U-shaped two-choice field can be used to measure animal’s behavioral responses to two contrasting or similar options, such as (i) a social target versus an inanimate object, (ii) a new stranger versus an earlier stranger, and (iii) a novel animal (a non-mate) versus a cage-mate (a familiar animal). We describe detailed procedures for sociability tests for the above three behavioral test paradigms based on the U-shaped two-choice field.
Keywords: Social interaction Social recognition Social novelty Social preference
Background
Sociability is the behavioral disposition of being sociable with others. Sociability is defective in psychiatric disorders including depression, autism and schizophrenia. Defective sociability is regarded to be an important behavioral symptom representing a state of psychiatric illness (Hirschfeld et al., 2000; American Psychiatric Association., 2013; Green et al., 2015; Barak and Feng, 2016). Animal models have been used to investigate the neural mechanisms of sociability, as well as the neuropathological mechanisms of psychiatric illness. A variety of sociability tests have been developed to measure specific aspects of social behaviors of experimental animals, such as social interactions (Moy et al., 2004; Berton et al., 2006; McFarlane et al., 2008; Silverman et al., 2010), social communication using olfactory and visual cues or vocalization (Arakawa et al., 2008; Radyushkin et al., 2009; Scattoni et al., 2009; Yang and Crawley, 2009), social novelty preference and social memory (Moy et al., 2004; Kim et al., 2017a; Lee et al., 2017).
A sociability test using a U-shaped two-choice field was developed to read out behavioral states of sociability in mice (Seo et al., 2012). The U-shaped two-choice field is easily set up by partitioning an open field with a wall to the central point, so that the two symmetrical rectangular fields, each containing closed and open square zones, form a ‘U-shaped’ two-choice field (Park et al., 2014; Kim et al., 2015). Here, we describe how a U-shaped two-choice field can be used to measure animal’s behavioral response to two distinctive or contrasting options, namely (i) a social target versus an inanimate object or empty environment, (ii) an earlier stranger versus a new stranger, and (iii) a cage-mate (familiar one) versus a non-mate (unfamiliar one) (Seo et al., 2012; Park et al., 2014; Kim et al., 2015; 2016, 2017a, 2017b; Kim and Han, 2016a, 2016b, 2016c; Choi et al., 2015). Detailed procedures and the utility of the different sociability tests based on the U-shaped two-choice field are described below.
Materials and Reagents
Latex examination gloves (Alliance: LGPF, Safeplus®)
Paper towels
50-ml polypropylene conical tubes
C57BL/6 mice
Note: Animals are described in detailed in Steps A1 and A2.
70% ethanol
Note: The 70% ethanol and paper towels are used to remove mouse excrement from the floor and walls of the U-shaped field and grid cages before the start of each behaviour test sessions.
Equipment
Two circular grid cages (Figure 1A)
Two circular grid cages (12 cm in diameter x 33 cm in height) made of tungsten wire.
Notes:
The grid cage needs to be high enough to prevent subject mice from climbing.
It is helpful to cover the upper half of the grid cage with an overhead projector (OHP) film to prevent the subject mouse from climbing, and to add a white opaque partitioning in the middle of the grid cage to block the target mouse from climbing (Figure 1A).
The U-shaped two-choice field (Figures 1B and 1C)
The U-shaped two-choice field is a modified open field (45 cm in width x 45 cm in depth x 35 cm in height) that is partially partitioned with a wall (20 cm in width x 35 cm in height) to the central point, so that a ‘U-shaped’ field that contains two closed quadrants and two open quadrants of the same size is formed (Figures 1B and 1C).
Notes:
The U-shaped two-choice field is a modified open field made of cream colored FOAMEX panel (1 cm in thickness) (Expanded PVC; LG Ltd., Republic of Korea).
The floor and walls of the U-shaped two-choice field are made of the same type of FOAMEX panel described above.
Alternatives for the walls and floor might be workable, but reflective plastic materials or uncoated veneer plywood is not recommended. The floor should not be slippery.
The U-field two-choice test is performed in an isolated room in the absence of potentially disrupting environmental factors. If an enclosed spacious room is not available, we recommend padding the outside walls of the U-shaped field with Styrofoam boards.
The behavior test room is lit with 2 or 4 indirect lighting sources to achieve 20 lux on the floor of the U-shaped field.
Figure 1. Photos showing the grid cages and U-shaped two-choice field. A. The circular grid cage. The upper half of the grid is covered with a transparent film. A white, opaque-circular partitioning is added in the middle of the grid cage. B. The open field is partially partitioned with a wall projecting to the center. C. A grid cage is placed in each outer corner of the closed quadrants making up the U-shaped two-choice field.
Brightness-adjustable indirect light sources
Digital Lux meter (TES Electrical Electronic, catalog number: TES-1350A )
White noise generator (HDT Korea, Genius mate)
Sound level meter (TES Electrical Electronic, catalog number: TES-1330A )
Stainless steel large forceps (20 cm in length) (Fine Science Tools, catalog number: 11000-20 ) with protective silicone tip guards (optional)
Automatic recording systems
A computerized video tracking system (SMART, Panlab, Spain) and PC
CCD camera (Samsung, catalog number: SDC-410 ) for video tracking
Webcam camera (Logitech, catalog number: C210 ) (used as a supplemental and independent recording system)
Software
A computerized video tracking system (SMART, Panlab, Spain)
GraphPad PRISM 6.0 software (GraphPad Software. Inc., San Diego, CA, USA) for data analysis
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lee, E., Park, J., Lee, Y. and Han, P. (2018). Sociability and Social Novelty Preference Tests Using a U-shaped Two-choice Field. Bio-protocol 8(10): e2853. DOI: 10.21769/BioProtoc.2853.
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Category
Neuroscience > Behavioral neuroscience > Cognition
Neuroscience > Nervous system disorders > Animal model
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2,854 | https://bio-protocol.org/exchange/protocoldetail?id=2854&type=0 | # Bio-Protocol Content
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Peer-reviewed
Intra-amniotic Injection of Mouse Embryos
LC Liyuan Cui*
PZ Peng Zou*
QM Qingshuo Meng
LL Lu Lu
JZ Jiayi Zhang
*Contributed equally to this work
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2854 Views: 6185
Reviewed by: Nolwenn PasquetAnca Savulescu
Original Research Article:
The authors used this protocol in Jun 2017
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Abstract
Recent outbreaks of infectious neuro-developmental diseases such as congenital Zika syndrome - have led to a demand for prognosis data from animal models. We developed an intra-amniotic injection mice model that allows Zika virus (ZIKV) infected mice to grow to puberty. In this system, ZIKV is injected into the amniotic fluid of pregnant mice and infected embryos thereafter. ZIKV-infected mice show several symptoms of clinical ‘congenital Zika syndrome’, including decreased brain volume and mis-laminated retina. We also evaluated several behavioral functions of these ZIKV-infected mice, for example, after the mice reach puberty, they have visual and motor defects. This technique can be used to screen and evaluate drug candidates and may help evaluate the prognosis of infectious neuro-developmental diseases.
Keywords: Intra-amniotic Embryos Virus Infection
Background
It has been more than sixty years since the first human case of ZIKV infection was reported but in 2007 only 14 human cases of ZIKV infection had been recorded. Prognosis data have lagged far behind the recent outbreak of ZIKV in 2015. There is some evidence that mouse models may be effective for prognosis studies because previous reports have proved neurovirulence of ZIKV in mice. Therefore, various methods of ZIKV infection including intravenous injection, intraperitoneal injection, foot-pad injection and brain injection have been used to study congenital Zika syndrome in individuals during the embryonic period or infancy. The intra-amniotic injection model presented here has two advantageous features. First, wild-type mice (C57 BL/6J) can be used and studies are not limited to mice with immunologic deficiencies. Second, ZIKV infected mice can grow into puberty, which is beneficial to studies trying to evaluate prognosis.
Materials and Reagents
Glass pipettes (World Precision Instrument, catalog number: 4878 )
Sterile gauze (Winner Medical Group, catalog number: 016935 )
Cotton ball (Winner Medical Group, catalog number: 50401050 )
1 ml Syringe (KDL, catalog number: 60017031 )
50 ml centrifuge tube (Corning, catalog number: 430828 )
Dropper (Shanghai Baiqian Biotechnology, catalog number: J00082 )
Suture needle (Ningbo Medical Needle Co., LTD, 7/0)
Pregnant mouse (E15) (Shanghai Yison Biotechnology Company, strain: C57 BL/6J)
75% alcohol (Sinopharm Chemical Reagent, catalog number: 80176960 )
Oxygen (Shanghai Lvmin Gas company, purity > 99%)
Cyanoacrylate (Pattex®, catalog number: PSK12CT-2 )
Isoflurane (RWD Life Science, catalog number: R510-22 )
Iodophor (Shanghai Likang Disinfectant Hi-Tech, catalog number: 310100 )
Lidocaine (MP Biomedicals, catalog number: 190111 )
Ampicillin (Inalco, catalog number: 1758-9314 )
Dulbecco’s modified Eagle’s medium (DMEM) (Corning, catalog number: 10-013-CV )
10% fetal bovine serum (FBS) (Biological Industries, catalog number: 04-001-1ACS )
Pen-Strep Solution (Penicillin: 10,000 U/ml, Streptomycin: 10 mg/ml) (Biological Industries, ISRAEL, catalog number: 03-031-1B )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (Sigma-Aldrich, Fluka, catalog number: 71645 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
0.1 M Phosphate buffer solution (PBS) (see Recipes)
Ampicillin solution (see Recipes)
Culture medium (see Recipes)
1% lidocaine solution (see Recipes)
Equipment
Flaming/brown micropipette puller (Sutter Instrument, model: P-97 )
Anesthesia machine (RWD Life Science, catalog number: R610 )
Water bath (Jinghong Experimental Equipment, model: XMID-8222 )
Tweezers (VETUS, catalog number: ST-11 )
Scissors (RWD Life Science, catalog number: S12003-09 )
Shaver (Codos, catalog number: KP-3000 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Cui, L., Zou, P., Meng, Q., Lu, L. and Zhang, J. (2018). Intra-amniotic Injection of Mouse Embryos. Bio-protocol 8(10): e2854. DOI: 10.21769/BioProtoc.2854.
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Category
Cell Biology > Cell Transplantation > Embryo Transplant
Microbiology > Microbe-host interactions > Virus
Neuroscience > Nervous system disorders > Animal model
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2,855 | https://bio-protocol.org/exchange/protocoldetail?id=2855&type=0 | # Bio-Protocol Content
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Peer-reviewed
Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies
CA Chie Aoki-Utsubo*
MC Ming Chen*
Hak Hotta
*Contributed equally to this work
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2855 Views: 12072
Edited by: Vamseedhar Rayaprolu
Reviewed by: Balaji Olety AmaranathKathrin Sutter
Original Research Article:
The authors used this protocol in Nov 2017
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Abstract
In a narrow definition, virucidal activity represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of the viral particles. The viral infectivity can be measured in cell culture system by means of plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID50) assay, etc. Morphologically, disruption of viral particles can be demonstrated by negative staining electron microscopic analysis of viral particles. In this article, we describe methods to assess virucidal activity in a broad definition.
Keywords: Virucidal activity Neutralizing activity Viral particle Antiviral substance Antibody Viral infectivity assay Negative staining electron microscopic analysis
Background
Viruses are small intracellular parasites that hijack host cell machinery to replicate their own genome. At the initial step of the viral life cycle, infectious viral particles attach (bind) to particular host proteins, called viral receptors, on the surface of the target cells, followed by viral penetration (internalization and/or fusion) into intracellular compartments of the host cells, where the subsequent steps of the viral life cycle proceed to produce progeny virions (Scheel and Rice, 2013).
Virucidal activity in a narrow definition represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to interact with and functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of viral particles, as in the case of antibody-mediated neutralization.
We have recently reported that an isoform of secreted phospholipase A2 obtained from snake venom (Chen et al., 2017) and a peptide from scorpion venom (El-Bitar et al., 2015) possess strong virucidal activity against viruses that belong to the family Flaviviridae by targeting the lipid bilayer of the viral envelope, which is acquired from the endoplasmic reticulum membrane of the host cells. It was also reported that one of the host defense peptides from the skin of the South Indian frog has a strong virucidal activity against H1 hemagglutinin-bearing human influenza virus by targeting the conserved stalk of H1 hemagglutinin (Holthausen et al., 2017). In this article, we describe a number of useful methods by which to measure virucidal activity in a broad definition, such as plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID50) assay and negative staining electron microscopic analysis.
Materials and Reagents
Disposable tips
10 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3510-05 )
200 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3900 )
1 ml capacity (FUKAEKASEI and WATSON, catalog number: 110-502C )
100 mm culture dish (Corning, Falcon®, catalog number: 353003 )
6-well culture plate (Corning, Falcon®, catalog number: 353046 )
12-well culture plate (Corning, Falcon®, catalog number: 353043 )
24-well culture plate (Corning, Falcon®, catalog number: 353047 )
96-well culture plate (Corning, Falcon®, catalog number: 353072 )
1.5 ml microcentrifuge tube (FUKAEKASEI and WATSON, catalog number: 131-715C )
15 ml tube (Corning, Falcon®, catalog number: 352196 )
Cover slip (13 x 13 mm; Matsunami Glass, catalog number: C013001 )
Microscope slide (Matsunami Glass, catalog number: S2215 )
Disposable serological pipette
1 ml capacity (Corning, Falcon®, catalog number: 356521 )
5 ml capacity (IWAKI, catalog number: 7153-005 )
10 ml capacity (IWAKI, catalog number: 7154-010 )
Filter paper (ATTO, catalog number: CB-06A-20A )
Viruses (Chen et al., 2017):
Hepatitis C virus (HCV, J6/JFH-1 strain)
Dengue virus (DENV, Trinidad 1751 strain)
Japanese encephalitis virus (JEV, Nakayama strain)
Influenza A virus (FLUAV, A/Udorn/307/72[H3N2])
Sendai virus (SeV, Fushimi strain)
Herpes simplex virus type 1 (HSV-1, CHR3 strain)
Coxsackievirus B3 (CV-B3, Nancy strain)
Vesicular stomatitis New Jersey virus (VSNJV)
Sindbis virus (SINV)
Encephalomyocarditis virus (EMCV, DK-27 strain)
Huh7it-1 cells (Apriyanto et al., 2016)
Note: Huh7it-1 cells are susceptible to all viruses described above (HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3, VSNJV, SINV and EMCV).
Vero cells (ATCC, catalog number: CCL-81 )
Note: Vero cells are susceptible to all viruses described above (HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3, VSNJV, SINV and EMCV).
Antibodies
Rabbit polyclonal antibody against DENV PrM (Gene Tex, catalog number: GTX128093 )
Mouse monoclonal antibody against DENV type 2 (3H5; Hotta et al., 1984)
UV-inactivated anti-HCV human serum (Bungyoku et al., 2009)
Anti-HCV E2 neutralizing antibody #55 (Shimizu et al., 2013)
Rabbit antiserum against CV-B3 (DENKA SEIKEN, catalog number: 300638 )
Rabbit antiserum against FLUAV (Shimizu et al., 1985)
Rabbit antiserum against SeV (Hayashi et al., 1991)
Rabbit antiserum against HSV-1 (Hayashi et al., 1986)
FITC-conjugated goat anti-human IgG (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 104AG )
Alexa Flour488-conjugated goat anti-mouse IgG (Thermo Fisher Science, catalog number: A-11001 )
Alexa Flour488-conjugated goat anti-rabbit IgG (Thermo Fisher Science, catalog number: A-11008 )
High glucose Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, catalog number: 044-29765 )
Phospholipase A2 from Naja mossambica snake venom (Sigma-Aldrich, catalog number: P7778 ) (Chen et al., 2017)
Trypsin-EDTA solution (Wako Pure Chemical Industries, catalog number: 209-16941 )
Crystal violet (Wako Pure Chemical Industries, catalog number: 038-04862 )
MEM with non-essential amino acids (Thermo Fisher Science, GibcoTM, catalog number: 10370021 )
Fetal bovine serum (FBS; Biowest, catalog number: S1820 )
Penicillin-Streptomycin solution (Wako Pure Chemical Industries, catalog number: 168-23191 )
Methyl Cellulose 4000 (Wako Pure Chemical Industries, catalog number: 136-02155 )
4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries, catalog number: 163-20145 )
Formaldehyde solution (AppliChem, catalog number: A3592,0500 )
Gram Hacker’s Stain Solution I (MUTO PURE CHEMICALS, catalog number: 41162 )
Triton X-100 (Wako Pure Chemical Industries, catalog number: 169-21105 )
Bovine serum albumin (BSA; Wako Pure Chemical Industries, catalog number: 015-21274 )
Hoechst 33342 solution (Thermo Fisher Scientific, Molecular Probes, catalog number: H3570 )
Vectashield mounting solution (Vector Laboratories, catalog number: H-1000 )
Formvar-coated nickel grid (Electron Microscopy Sciences, catalog number: FF200-Ni )
2% phosphotungstic acid (Wako Pure Chemical Industries, catalog number: 582-66852 )
Sodium chloride (NaCl; Wako Pure Chemical Industries, catalog number: 191-01665 )
Potassium chloride (KCl; Wako Pure Chemical Industries, catalog number: 163-03545 )
Disodium Hydrogen Phosphate (Na2HPO4·12H2O, NACALAI TESQUE, catalog number: 31722-45 )
Potassium phosphate monobasic (KH2PO4; Wako Pure Chemical Industries, catalog number: 169-04245 )
Complete medium for cell culture (see Recipes)
10x phosphate-buffered saline (PBS[-]) (see Recipes)
Overlay medium (see Recipes)
Equipment
Micropipette (Gilson, P20, P200, P1000)
P20 (Gilson, catalog number: F123600 )
P200 (Gilson, catalog number: F123601 )
P1000 (Gilson, catalog number: F123602 )
Multichannel micropipette (10-100 μl) (Eppendorf, catalog number: 3125000036 )
Hemocytometer chamber (e.g., Erma, catalog number: 03-303-1 )
Biosafety cabinet (e.g., PHC, model: MHE-S1301A2 )
CO2 incubator (e.g., PHC, model: MCO-20AIC )
Autoclave (e.g., TOMY DIGITAL BIOLOGY, model: SX-500 )
Refrigerated tabletop centrifuge (e.g., Eppendorf, model: Centrifuge 5424 )
Vortex (e.g., Scientific Industries, model: Vortex-Genie 2 )
-80 °C freezer (e.g., PHC, model: MDF-384 )
Inverted microscope (e.g., Olympus, model: CKX53 )
Fluorescent microscope (e.g., ZEISS, model: Axio Vert. A1 )
Multilabel Plate Counter (PerkinElmer, model: 1420 ALBOSX )
Transmission electron microscope (Hitachi, model: HT7700 TEM )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Aoki-Utsubo, C., Chen, M. and Hotta, H. (2018). Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies. Bio-protocol 8(10): e2855. DOI: 10.21769/BioProtoc.2855.
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Category
Microbiology > Antimicrobial assay > Antiviral assay
Cell Biology > Cell-based analysis > Viral infection
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2,856 | https://bio-protocol.org/exchange/protocoldetail?id=2856&type=0 | # Bio-Protocol Content
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Enzymatic Activity Assay for Invertase in Synechocystis Cells
XT Xiaoming Tan
KS Kuo Song
XL Xuefeng Lu
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2856 Views: 6839
Original Research Article:
The authors used this protocol in Jun 2017
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Jun 2017
Abstract
Invertase can catalyze the hydrolysis of sucrose, and is widely distributed in cells of cyanobacteria and plants. Being responsible for the first step for sucrose metabolism, invertase plays important physiological roles and its enzymatic activity is frequently needed to be determined. All the methods for determination of the invertase activity are dependent on detection of the glucose product generated by the invertase. Here we describe an ion chromatography based protocol of our laboratory for determination of cyanobacterial intracellular invertase activity.
Keywords: Cyanobacteria Synechocystis Invertase enzymatic activity Sucrose Ion chromatography
Background
Invertase and sucrose play important physiological roles in cyanobacteria (Curatti, et al., 2008; Kolman et al., 2015) and higher plants (Vargas et al., 2003; Vargas et al., 2010). Invertase (EC 3.2.1.26) can catalyze the sucrose degradation into glucose and fructose. Due to this characteristic of the invertase, any methods which could be used for the determination of glucose or fructose would be theoretically used for the invertase enzymatic activity assay. In fact, most of the invertase enzymatic activity assays are based on the detection of the generated glucose product.
Some companies have developed several kits for the invertase activity assay, for instances, ab197005 from abcam (USA), KA1629 from Novus Biologicals (USA), MAK118 from Sigma-Aldrich (USA). By using these kits, the glucose product generated from the invertase reactions would be oxidized and determined by a colorimetric (570 nm) or fluorimetric method (λem/ex = 585/530 nm). In other methods, the amount of reducing sugar liberated by invertase was measured by coupling hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase, while the resulting NADPH was further spectrophotometrically determined at 340 nm (Vargas et al., 2003). Ion chromatography could directly detect various sugars including glucose, fructose, sucrose (Du et al., 2013) and glucosylglycerol (Tan et al., 2015). Compared with the spectrophotometer-based methods for sugar determinations, ion chromatography (IC) could be more beneficial for analyzing the enzymatic mixtures containing multiple compounds, especially for the enzymatic assay of cell crude extracts. By using ion chromatography, the sucrose consumption and the glucose production could be shown at the same time in the case of the invertase enzymatic assay, which would provide complete information for the enzymatic assays.
Here, we report our recent IC-based protocol for determination of the invertase activity in cyanobacterial cells.
Materials and Reagents
Pipette tips
2 ml tubes (Cypress, China)
10 ml conical tubes (Kangjian, China)
1 ml syringe (Jianshi, China)
Syringe membrane filters, 0.22 μm (Jinteng, China)
Synechocystis sp. PCC 6803 (Tan et al., 2011)
Milli-Q water (Millipore, Germany)
Glass beads (Sigma-Aldrich, catalog number: G9018-250G )
Fructose standard (Sinopharm Chemical Reagent, catalog number: 63003034 )
Glucose standard (Sinopharm Chemical Reagent, catalog number: 10010518 )
Liquid nitrogen
200 mM NaOH (prepared with Milli-Q water)
Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10013018 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sinopharm Chemical Reagent, catalog number: 20011160 )
Critic acid monohydrate (C6H8O7·H2O) (Sinopharm Chemical Reagent, catalog number: 10007118 )
Ferric ammonium citrate ((NH4)3FeC12H10O14/C6H8O7·xFe3·yNH3) (Sinopharm Chemical Reagent, catalog number: 30011428 )
EDTA·2Na·2H2O (Sinopharm Chemical Reagent, catalog number: 10009717 )
Sodium carbonate (Na2CO3) (Sinopharm Chemical Reagent, catalog number: 10019260 )
Boric acid (H3BO3) (Sinopharm Chemical Reagent, catalog number: 10004818 )
Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sinopharm Chemical Reagent, catalog number: 20026118 )
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10024018 )
Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sinopharm Chemical Reagent, catalog number: 10019818 )
Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sinopharm Chemical Reagent, catalog number: 10008218 )
Cobalt(II) chloride hexahydrate (CoCl2·6H2O) (Sinopharm Chemical Reagent, catalog number: 10007216 )
Sodium nitrate (NaNO3) (Sinopharm Chemical Reagent, catalog number: 10019918 )
Potassium dihydrogen phosphate (KH2PO4) (Sinopharm Chemical Reagent, catalog number: 10017618 )
Dipotassium hydrogen phosphate trihydrate (K2HPO4∙3H2O) (Sinopharm Chemical Reagent, catalog number: 10017518 )
Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
Isopropanol (Sinopharm Chemical Reagent, catalog number: 40064360 )
BG11 medium (see Recipes)
100 mM potassium phosphate buffer (pH 7.0) (see Recipes)
100 mM PMSF (see Recipes)
Equipment
50 ml flasks
Pipettes (Eppendorf, Germany)
Shaker (Taicang Huamei, model: THZ-701B )
Water bath (Shanghai Yarong, model: B-260 )
Centrifuge (Beckman Coulter, model: Microfuge® 22R )
-20 °C freezer (Haier, model: BCD-219D )
Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
Thermal Cycler for PCR (Bio-Rad Laboratories, model: T-100 )
Ion chromatography (Thermo Fisher Scientific, Thermo ScientificTM, model: DionexTM ICS-5000+ )
DionexTM CarboPacTM PA10 analytical column (4 x 250 mm, Thermo Fisher Scientific, model: DionexTM CarboPacTM PA10 )
Software
ChromeleonTM software (Thermo Fisher Scientific, Thermo ScientificTM, version 6.80; catalog number: CHROMELEON6)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Tan, X., Song, K. and Lu, X. (2018). Enzymatic Activity Assay for Invertase in Synechocystis Cells. Bio-protocol 8(10): e2856. DOI: 10.21769/BioProtoc.2856.
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Category
Microbiology > Microbial biochemistry > Carbohydrate
Microbiology > Microbial metabolism > Carbohydrate
Biochemistry > Protein > Activity
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2,857 | https://bio-protocol.org/exchange/protocoldetail?id=2857&type=0 | # Bio-Protocol Content
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Ectopic Gene Expression in Macrophages Using in vitro Transcribed mRNA
Pallavi Chandra
JP Jennifer A. Philips
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2857 Views: 6994
Edited by: Alka Mehra
Reviewed by: Gal HaimovichRan Chen
Original Research Article:
The authors used this protocol in Oct 2017
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The authors used this protocol in:
Oct 2017
Abstract
Macrophages are immune cells that contribute to host defense through various mechanisms including phagocytosis and antigen presentation. Their antimicrobial capacity is subverted by clinically important intracellular pathogens such as Mycobacterium tuberculosis. The study of host-pathogen interactions using these cells is therefore of considerable interest. Such studies often seek to express tagged proteins to characterize their activities, localizations, and protein-protein interactions. Here, we describe a robust method for transient protein expression in macrophages using mRNA lipoplex transfections.
Keywords: mRNA transfection Macrophage Protein expression
Background
Typical methods to achieve protein expression, including transfecting DNA using cationic polymers, nucleofection, and viral transduction (Zhang et al., 2009), are particularly difficult in macrophages, as these cells have a potent immune response to various danger signals such as cytosolic DNA. Therefore, conventional methods for exogenous gene delivery result in poor transfection efficiency and cell death. We reasoned that transfecting mRNA, instead of DNA, would be a better alternative to achieve protein expression in macrophages, as suggested by recent reports (Van De Parre et al., 2004; McLenachan et al., 2013). We were able to achieve high transfection efficiencies without loss of macrophage viability (Koster et al., 2017). Our method does not require expensive equipment and can be adapted for expressing exogenous and endogenous proteins.
Materials and Reagents
μ-Plate 96 Well (ibidi, catalog number: 89626 )
Sterile RNase-free microfuge tubes (Thermo Fischer Scientific, catalog number: AM12400 )
DNase and RNase free sterile pipet tips (e.g., Sorenson Bioscience, catalog number: 10350 )
Gloves (e.g., powder free nitrile gloves, MICROFLEX, catalog number: XC-310 )
Macrophages
Macrophage cell types that can be used are cell lines such as RAW 264.7 (ATCC, catalog number: TIB-71 ) and primary macrophages such as C57BL/6 bone marrow-derived macrophages (BMDMs).
Cell culture medium
RAW 294.7 cell lines were maintained in Dulbecco Modified Eagle Medium (DMEM, Thermo Fisher Scientific, GibcoTM, catalog number: 11965 ) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 ).
CleanCapTM EGFP mRNA (TriLink BioTechnologies, catalog number: L-7601 )
TransIT®-mRNA Transfection Kit (Mirus Bio, catalog number: MIR 2250 )
OptiMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
RNaseZapTM (Sigma-Aldrich, catalog number: R2020 )
mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific, AmbionTM, catalog number: AM1345 )
MEGAClearTM Transcription Clean-Up Kit (Thermo Fisher Scientific, AmbionTM, catalog number: AM1908 )
Molecular biology grade water (Corning, catalog number: 46-000-CI )
Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16140071 )
Penicillin and streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
BglII restriction endonuclease (New England Biolabs, catalog number: R0144S )
NEBuffer 3.1 (New England Biolabs, catalog number: B7203S )
Antarctic Phosphatase Reaction Buffer (New England Biolabs, catalog number: B0289S )
Antarctic Phosphatase (New England Biolabs, catalog number: M0289S )
QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
GatewayTM pcDNATM-DEST47 vector (Thermo Fischer Scientific, catalog number: 12281010 )
Agarose (LE Agarose, GeneMate, catalog number: E-3120-500 )
10x MOPS buffer (Fischer Scientific, catalog number: BP2900500 )
Ethidium bromide (Sigma-Aldrich, catalog number: E1510 )
RNA Millennium Markers (Thermo Fisher Scientific, AmbionTM, catalog number: AM7150 )
RNA gel loading dye (Thermo Fischer Scientific, catalog number: R0641 )
Formaldehyde solution (37%, Sigma-Aldrich, catalog number: 252549 )
Plasmid linearization reaction mix (see Recipe 1)
EGFP mRNA transfection reaction mix (see Recipe 3)
Poly(A) tailing reaction mix (see Recipe 4)
RNA denaturing gel electrophoresis (see Recipe 5)
mRNA in vitro transcription reaction mix (see Recipe 6)
DMEM complete medium (see Recipe 7)
Equipment
P20 Pipetman (Gilson, catalog number: F123600 )
P200 Pipetman (Gilson, catalog number: F123601 )
P1000 Pipetman (Gilson, catalog number: F123602 )
Multi-channel pipette (Eppendorf, catalog number: 3125000044 )
Tissue culture CO2 incubator
Tissue culture hood
Water bath
-20 °C freezer
Epifluorescence microscope (e.g., Nikon, model: Eclipse Ti-E , equipped with 60x; Plan-Apochromat, NA 1.4 oil immersion objective, Ti Z drive, high-resolution monochrome charge-coupled device (CCD) digital camera; Photometric Cool SNAP HQ2 and appropriate filter sets for DAPI, FITC and TexasRed channel)
Eppendorf Thermomixer® C (Eppendorf, model: ThermoMixer® C , catalog number: 5382000023)
NanoDrop Spectrophotometer
Agarose gel electrophoresis system
Software
Nikon Imaging Software-Elements Advanced Research (NIS-Elements) version 4.40
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chandra, P. and Philips, J. A. (2018). Ectopic Gene Expression in Macrophages Using in vitro Transcribed mRNA. Bio-protocol 8(10): e2857. DOI: 10.21769/BioProtoc.2857.
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Category
Molecular Biology > RNA > mRNA translation
Molecular Biology > RNA > Transfection
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2,858 | https://bio-protocol.org/exchange/protocoldetail?id=2858&type=0 | # Bio-Protocol Content
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Osteoblast Sorting and Intracellular Staining of CXCL12
WW Weihuan Wang
GM Gurnoor Majhail
CL Cui Lui
LZ Lan Zhou
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2858 Views: 6434
Edited by: Nicoletta Cordani
Reviewed by: Abhijit Kale
Original Research Article:
The authors used this protocol in Mar 2016
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Mar 2016
Abstract
Osteoblasts are bone marrow endosteum-lining niche cells playing important roles in the regulation of hematopoietic stem cells by secreting factors and cell adhesion molecules. Characterization of primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone. Immunophenotyping and flow-based analysis of long bone osteoblasts offer a simplified and rapid approach to characterize osteoblasts. We describe a modified procedure of isolating mouse bone marrow osteoblastic cells based on cell surface immunophenotyping. The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are expressed by osteoblasts and bone marrow stroma cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches (Sugiyama et al., 2006) and for supporting leukemia initiating cell activity (Pitt et al., 2015). Here we describe the procedure of intracellular staining of CXCL12.
Keywords: Bone marrow niche HSC Osteoblast CXCL12 Intracellular staining
Background
The bone marrow niche is a highly organized microenvironment with stroma cells that engage in direct cell-cell interaction with hematopoietic stem cells (HSC) that regulate HSC quiescence, differentiation, and mobilization (Anthony and Link, 2014; Mendelson and Frenette, 2014; Morrison and Scadden, 2014). Multiple cell types in HSC niche may contribute to niche functional support in distinct but maybe overlapping ways. These cells include but are not limited to osteoblasts, osteoclasts, CXCL12-abundant reticular (CAR) cells, Nestin+ stroma cells, leptin receptor+ (LepR+) stroma cells, endothelial cells, macrophages, megakaryocytes, neuronal, and the non-myelinating Schwann cells. Most HSCs are found in the trabecular region of bone marrow, suggesting an important HSC supporting role of the endosteum as well as factors made by osteoblasts and other cells in the endosteum (Kiel et al., 2005; Lo Celso et al., 2009). The majority of long-term HSCs are located in close vicinity of the sinusoid in close contact with LepR+ and CXCL12high niche cells, indicating a perivascular niche composed by endothelial or perivascular cells (Kiel et al., 2005; Sugiyama et al., 2006; Acar et al., 2015). In addition to the perivascular niche associated with sinusoid, mesenchymal cells that surround arterioles in the bone marrow are also important for the maintenance of quiescent HSCs (Kunisaki et al., 2013). Osteoblasts are specialized endosteum-lining cells that are terminally differentiated products of mesenchymal stem cells. Characterization of murine primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone and retrospective functional analysis (Bakker and Klein-Nulend, 2011). However, culture-based analysis has been complicated by the heterogeneity of the tissue. Immunophenotyping of murine osteoblasts based on defined CD markers is a rapid and prospective approach of phenotypic analysis of osteoblasts in various disease processes. Isolated osteoblasts through flow-based sorting are especially suitable for downstream applications such as gene expression analysis.
The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are highly expressed by CAR cells but also by osteoblasts and endothelial cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches (Sugiyama et al., 2006). CXCL12 in the vascular niche has also been shown play a critical role in supporting leukemia-initiating cell (LIC) activity (Pitt et al., 2015). Using a mouse T-ALL model, we reported that leukemia development was accompanied by the drastic suppression of the endosteum-lining osteoblast population. We further showed that aberrant Notch activation negatively regulates the expression of CXCL12 and osteoblastic progenitor differentiation. Here we describe the procedure of sorting mouse bone marrow osteoblastic cells and the procedure of staining intracellular CXCL12 (Wang et al., 2016).
Materials and Reagents
Gauze sponges (Fisher Scientific, FisherbrandTM, catalog number: 22-415-468 )
21 G needles
3 ml syringes
70 μm strainer (Fisher Scientific, FisherbrandTM, catalog number: 22-363-548 )
1.5 ml microcentrifuge tube (NEST Biotechnology, catalog number: 615601 ), 15 ml tubes (BioExpress, GeneMateTM, catalog number: C-3394-1 ), and 50 ml conical tubes (BioExpress, GeneMateTM, catalog number: C-3394-4 )
Other antibodies:
APC-anti-CD31 (Thermo Fisher Scientific, eBioScienceTM, catalog number: 17-0311-82 )
PE-anti-CD51 (BD, BD PharmingenTM, catalog number: 551187 )
PE-Cy7-anti-CD45 (BD, BD PharmingenTM, catalog number: 552848 )
Biotin-Sca1 (BD, BD PharmingenTM, catalog number: 553334 )
Streptavidin APC-Cy7 (BD, BD PharmingenTM, catalog number: 554063 )
CXCL12 detection antibodies: H/M CXCL12/SDF-1 Fluorescein (FITC) MAb (Clone 79018) (R&D Systems, catalog number: IC350F )
70% (v/v) ethanol
Hank’s Balanced Salt Solution (HBSS; 1x) (GE Healthcare, HycloneTM, catalog number: SH30588.02 )
BSA (Sigma-Aldrich, catalog number: A7906 )
Type I collagenase (Worthington, Lakewood, NJ)
DMEM (ATCC®, catalog number: 30-2002TM )
Fixation/Permeabilization Solution Kit with BD GolgiStopTM (BD, BD Cytofix/Cytoperm™ Plus, catalog number: 554715 )
HBSS staining buffer (see Recipes)
Equipment
Pipettes
Scissors
Tweezers
Curved forceps
Tabletop centrifuge (SORVALL Legend RT)
Hemocytometer
FACSAria I
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wang, W., Majhail, G., Lui, C. and Zhou, L. (2018). Osteoblast Sorting and Intracellular Staining of CXCL12. Bio-protocol 8(10): e2858. DOI: 10.21769/BioProtoc.2858.
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Category
Stem Cell > Adult stem cell > Hematopoietic stem cell
Cell Biology > Cell staining > Cell wall
Cell Biology > Cell-based analysis > Flow cytometry
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2,859 | https://bio-protocol.org/exchange/protocoldetail?id=2859&type=0 | # Bio-Protocol Content
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Rapid Screening and Evaluation of Maize Seedling Resistance to Stalk Rot Caused by Fusarium spp.
YS Yali Sun
XR Xinsen Ruan
LM Liang Ma
FW Fang Wang
XG Xiquan Gao
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2859 Views: 7374
Edited by: Zhibing Lai
Original Research Article:
The authors used this protocol in Aug 2007
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Aug 2007
Abstract
Corn stalk rot caused by Fusarium spp., a genus of soil-borne fungal pathogens, has become a major concern of maize production. This disease normally causes significant reduction of maize yield and quality worldwide. The field assay for identifying stalk rot resistance using adult plants is largely relying on large population, yet time-consuming, labor costs, and often influenced by environmental conditions. Therefore, a rapid and reliable assay for investigating maize stalk rot caused by Fusarium spp. is required for screening the resistant lines and functional study of maize resistance to this pathogen. We have developed a seedling assay to rapidly screen the resistant lines using 12-day to 2-week-old seedlings. The entire assay can be completed within approximately 16-18 days post seed germination, with inexpensive labor cost and high repeatability. This simple, rapid and reliable assay can be widely used for identifying the maize resistance to stalk rot caused by Fusarium spp. and other similar fungal pathogens.
Keywords: Corn stalk rot Disease resistance Fusarium spp. Seedling assay
Background
Maize is one of the most important staple food crops and energy plants worldwide. It has been becoming the No.1 crop species in China and worldwide regarding both yield and planting areas since 2012. However, corn stalk rot has become one of the most destructive diseases, which normally lead to significant yield loss and quality reduction of maize extensively (Oerke, 2006). The pathogens causing corn stalk rot mainly include the soil-borne fungal species, such as Fusarium graminearum, F. verticillioides, Colletotrichum graminicola, Pythium aphanidermatum, and Pectobacterium chrysanthemi. Infection of maize by F. graminearum (teleomorph Gibberella zeae Schw. Petch) normally causes Gibberella stalk rot with reddish-pink discoloration inside the stalk, and Gibberella ear rot with carcinogenic mycotoxins deoxynivalenol (DON) and zearalenone produced in mature corn kernels that are harmful to human and animals (Mesterhazy et al., 2012). Moreover, F. graminearum is also the major causal agent infecting other small grain cereal crops, such as wheat, leading to the well-known Fusarium head blight (FHB), which is one of the most serious diseases impacting wheat production in China and is endemic in many wheat-producing countries (Walter et al., 2010; Ma et al., 2017). F. verticillioides [=F. moniliforme J. Sheld.(sexual stage: G. moniliformis Wineland)], on the other hand, predominantly infect maize, resulting in Fusarium stalk rot and Fusarium ear rot, and producing Fumonisins, another type of mycotoxins in maize kernels (Presello et al., 2008). Thus, the increasing prevalence of mycotoxins produced by Fusarium spp., along with their direct impact on yield losses in many cereal-growing regions worldwide, has become one of the major concerns of much research.
In recent years, due to the rapid development and application of mechanization, the harvesting technology of corn grain by machines puts forward a high requirement of disease resistance to corn stalk rot. Currently, the field assay for identifying stalk rot resistance using adult plants is largely relying on large population, yet time-consuming, labor costs, and often influenced by environmental conditions. Therefore, a rapid, reliable and large-scale method for screening disease resistant lines and identifying the resistance to corn stalk rot is extremely in urgent, which can provide a great convenience for the researchers to rapidly excavate the elite genetic resources of maize. We have previously deployed a seedling assay to identify the resistance of maize lines to stalk rot caused by F. verticillioides (Gao et al., 2007). Here, we describe the detailed procedures of modified protocol of a seedling assay, which can be applied to evaluate the stalk rot not only caused by Fusarium spp., but also other fungal pathogens, such as C. graminicola.
Materials and Reagents
1.5 ml centrifuge tubes (Nanjing Qingke Biological Company)
50 ml centrifuge tubes (Nanjing Qingke Biological Company)
Long pot (20 cm tall x 5 cm diameter), hand-made with PVC tubes with a holed bottom cover
Parafilm (Bemis, catalog number: PM996 )
Press-in Saran Wrap (GLADR, Pressin Seal, 21.6 m x 30 cm), a type of sealing saran wrap with stickiness that can be pressed on the surface to seal tightly the object
Conical bottle (200 ml)
Paper towel (230 x 225 mm, Yong Li Yu Investment Company Limited, May Flower, catalog number: A18250S )
Cheese cloth (40S Warp x 40S Weft; 5 m x 1.2 m), made by combed cotton
Soil (Nanjing Shoude Company, nutrient soil: vermiculite = 2:1)
Syringe needle (Luer-Lok 20G, BD, catalog number: 309634 )
Plastic square trays (Purchased from market) (100 cm length x 80 cm width x 10 cm height)
Maize seeds (Inbred line: B73; select the similar size seeds with germinating rate > 90%)
Fungal strains: Fusarium graminearum (isolate: F0609); F. verticillioides (isolate: 7600)
Sterile ddH2O
Glycerin
PDA (Potato dextrose agar) (Qingdao Hope Bio-Technology, catalog number: HB0233 )
Tween-20 (SunShine Bio, catalog number: T0014 )
Mung Bean (Purchased from market)
PDA media (see Recipes)
Mung bean soup (see Recipes)
Equipment
Pipettes (Eppendorf, Research Plus, catalog numbers: 3120000020 , 3120000046 , 3120000062 )
Hemacytometer (0.100 mm, Hausser Scientific, catalog number: 3110 )
Water bath (Changzhou Nuoji Instrument, catalog number: HHS-11-1 )
Clean bench (AIRTECH, model: SW-CJ-1FD , catalog number: A16116317)
Constant temperature incubator (Ningbo Jiangnan Instrument Factory, catalog number: DNP-9162 )
Growth chamber (Ningbo Jiangnan Instrument Factory, model: RXM-508C-3 , catalog number: 17091559)
Centrifuge (Eppendorf, model: 5424 , catalog number: 5424FL570190)
Microscope (Olympus, catalog number: BX41 )
Autoclave
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sun, Y., Ruan, X., Ma, L., Wang, F. and Gao, X. (2018). Rapid Screening and Evaluation of Maize Seedling Resistance to Stalk Rot Caused by Fusarium spp.. Bio-protocol 8(10): e2859. DOI: 10.21769/BioProtoc.2859.
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Category
Plant Science > Plant immunity > Host-microbe interactions
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286 | https://bio-protocol.org/exchange/protocoldetail?id=286&type=0 | # Bio-Protocol Content
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Polysome Preparation, RNA Isolation and Analysis
Hailong Zhang
MZ Muxiang Zhou
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.286 Views: 27627
Original Research Article:
The authors used this protocol in Mar 2012
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Abstract
During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosomes can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.
Materials and Reagents
Cells (Neuroblastoma cell line SKN-SH)
Protease inhibitors (Sigma-Aldrich, catalog number: P8340-5ML )
DTT (Sigma-Aldrich, catalog number: 43815 )
Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 )
Heparin (Sigma-Aldrich, catalog number: H3149 )
Sucrose (Sigma-Aldrich, catalog number: S1888 )
Phenol/chloroform/isoamyl alcohol (Life Technologies, Invitrogen™, catalog number: 15593-031 )
Sodium acetate (Thermo Fisher Scientific, catalog number: S209-500 )
1x PBS
1x Trypsin-EDTA, 0.05% Trypsin/0.53 mM EDTA (Cellgro, catalog number: 25-052-CV )
RPMI-1640 (Hyclone, catalog number: SH30096.01 )
Tris-Base (Thermo Fisher Scientific, catalog number: BP-152-1 )
KCl (Thermo Fisher Scientific, catalog number: BP-366-500 )
MgCl2 (Sigma-Aldrich, catalog number: M-2393 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787-250ML )
DNAase/RNAase free Ethanol (Sigma-Aldrich, catalog number: E7023-500ML )
DNAase/RNAase free water (BioExpress, catalog number: UPW-1000 )
Ultracentrifuge tubes (14 x 89 mm) (Beckman Coulter, catalog number: 344059 )
Polysome extraction buffer (PEB) (see Recipes)
Sucrose solutions (see Recipes)
Equipment
BR-188 Density gradient fractionation system (Brandel)
Beckman optima L-70 ultracentrifuge (Beckman)
7500 Real-time PCR system (Applied Biosystems)
Boekel scientific orbitron rotator I, 115 V (Boekel Scientific)
Procedure
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Category
Molecular Biology > RNA > mRNA translation
Molecular Biology > RNA > RNA extraction
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2,860 | https://bio-protocol.org/exchange/protocoldetail?id=2860&type=0 | # Bio-Protocol Content
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A Method to Injure, Dissect and Image Indirect Flight Muscle of Drosophila
KC Kunal Chakraborty
KV K. VijayRaghavan
Rajesh Gunage
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2860 Views: 8633
Edited by: Leonardo Gaston Guilgur
Reviewed by: Thirupugal Govindarajan
Original Research Article:
The authors used this protocol in Oct 2017
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Abstract
Inducing an injury specifically to Drosophila flight muscles is a difficult task, owing to the small size of the muscles and the presence of the cuticle. The protocol described below provides an easy and reproducible method to induce injury in the Drosophila flight muscles.
Keywords: Drosophila flight muscles Injury Regeneration Insect satellite cells
Background
Muscles in vertebrates undergo regeneration, a process attributed to the Satellite cells, the resident stem cells. Our lab has recently shown that Drosophila flight muscles harbor stem cells similar to vertebrate satellite cells, namely insect satellite cells and show proliferation response to muscle injury (Chaturvedi et al., 2017). The ease of fly genetics and our method of inducing injury open up an opportunity to address relevant questions in the field of regenerative biology. We have standardized a protocol for injuring dorsal longitudinal muscles (DLMs) fibers with better precision, and this method can be used for investigating the repair mechanisms involved in muscles after the injury.
Materials and Reagents
Very thin paint brush
Minutien pins -Stainless Steel/0.1 mm Diameter (Minutien Pins) (Fine Science Tools, catalog number: 26002-10 )
Standard fly food media in glass vial (e.g., BDSC Cornmeal Food)
Standard Petri dish for immunostaining (e.g., FisherbrandTM Petri Dishes with Clear Lid, Thermo Fisher Scientific, catalog number: FB0875713 )
Adult Drosophila melanogaster
Cover slips (Thermo ScientificTM Gold SealTM Cover Slips) (Thermo Fisher Scientific, catalog number: 3306 )
Frosted micro slides, Size: 75 mm long x 25 mm wide, Thickness: 1.35 mm (Blue Star, BLUE STAR (FROSTED MICRO SLIDES))
Sharp razor blades (e.g., Gillette, 7 O'Clock Super Stainless BladesTM)
Double sided adhesive tape (Mario Tapes, catalog number: SP 110 )
Nail polish
(Optional) Liquid nitrogen
Ethanol (absolute for analysis EMSURE® ACS, ISO, Reag. Ph Eur) (Merck, catalog number: 1009831011 )
Sodium chloride (NaCl) (Sodium Chloride, Fisher BioReagents) (Fisher Scientific, catalog number: BP358-1 )
Potassium chloride (KCl) (Fisher Scientific, Potassium Chloride (Crystalline/USP/FCC), Fisher Chemical, catalog number: P330-500 )
Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, Sodium Phosphate Dibasic Anhydrous (USP), Fisher Chemical, catalog number: S375-500 )
Potassium phosphate monobasic (KH2PO4) (Potassium Phosphate Monobasic (Crystalline/Certified ACS)) (Fisher Scientific, Fisher Chemical, catalog number: P285-500 )
Triton X-100 (Sigma-Aldrich, catalog number: X100-500ML )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 05470-5G )
Phalloidin (1:500 in 1x PBS) (Alexa Fluor 488® phalloidin) (Thermo Fisher Scientific, catalog number: A12379 )
TOPRO-3-Iodide (1:1,000 in 1x PBS) (TO-PROTM-3 Iodide (642/661) - 1 mM Solution in DMSO) (Thermo Fisher Scientific, catalog number: T3605 )
VECTASHIELD Antifade Mounting Medium (Vector laboratories, Vectashield®, catalog number: H-1000 )
16% Paraformaldehyde (PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADE) (Electron Microscopy Sciences, catalog number: 15710 )
(Optional) Anti-myosin
(Optional) DAPI or Hoechst
70% (v/v) ethanol (see Recipes)
1x Phosphate buffered saline (PBS) (see Recipes)
16% Paraformaldehyde (PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADE) (Electron Microscopy Sciences, catalog number: 15710 ) (see Recipes)
Permeabilization solution (see Recipes)
Blocking solution (see Recipes)
Equipment
Stereo microscope (Olympus, model: SZX12 ) equipped with an imaging system (light source: Olympus KL1500 LCD, camera: QIClickTM CCD Camera, model: 01-QICLICK-R-F-CLR-12 , image acquisition software: Micromanager 1.4)
CO2 pad (e.g., FlyStuff flypad, Genesee Scientific, catalog number: 59-114 )
A Moria Nickel Plated Pin Holder (Moria Nickel Plated Pin Holder) (Fine Science Tools, catalog number: 26016-12 )
Forceps (Dumont #5 Forceps) (e.g., Fine Science Tool, catalog number: 11251-10 )
Scissor (Vannas Spring Scissors–2 mm Cutting Edge) (e.g., Fine Science Tools, catalog number: 15000-03 )
Laser Scanning Confocal Microscope Olympus FV1000 (FluoView® FV3000 Confocal Laser Scanning Microscope) (e.g., Olympus, model: FV3000 )
pH meter
Software
Image acquisition software:
Olympus Fluoview 1000 for Laser Scanning Confocal Microscope
Micromanager 1.4 for recording the video
Image processing software:
Fiji
GNU Image Manipulation Program (GIMP)
Procedure
Fly muscle injury
Take one Minutien pin with a pin holder as shown in Figure 1B.
Clean the pin with 70% v/v ethanol and then with distilled water and dry to avoid contamination. Repeat this procedure every time before pricking the fly with the pin.
Take 1-2 day old adult Drosophila and anesthetize them with CO2 on a CO2 pad (Figure 1A). Avoid taking more than 10-15 flies on a pad.
Note: Avoid overexposure of CO2 to the flies. Use of cold anesthesia is not suitable for this process as it might have unwanted cold injury effects. This can significantly alter the results.
Place the fly laterally as shown in Figure 1A under a stereo microscope and hold the thorax-abdomen junction gently with the light thin paint brush in one hand.
Note: Avoid use of excessive force while orienting and holding the flies for inducing injury.
While holding the fly, prick the thorax with the pin in PS (presutural) region as circled in Figure 1A. See the Video 1 as a reference.
Video 1. Injuring DLMs by a single pinprick at PS (presutural) region. For demonstration purpose, the fly was immobilized using a little drop of nail polish on a glass slide. During actual experiments use of any adhesive is unnecessary. As shown in the video, we make a single prick at PS (presutural) location highlighted with a red arrow (Also shown in Figure 1A). To minimize damage to surrounding tissue, it is crucial to be quick and strictly to avoid moving the pin once it pierces fly thorax. This video was acquired using Stereo microscope coupled with a camera (see Equipment section for technical details). For image acquisition, an open source software micromanager (μManager) has been used. For further reference see Edelstein et al., 2014. Images and videos were processed using Fiji.
Pricking by the pin should be at an angle of 45° to the A-P axis (anterior-posterior axis is the line running from head to abdomen that divides the animal with bilateral symmetry) so that only DLMs get injured (Figure 1C).
Alternative step: Before pricking the thorax, the pin can be cooled by liquid nitrogen by dipping for a brief moment.
The thorax should be injured with a pin by inserting ~0.5 mm so that only one of the hemithorax get injured (Figure 1D). This will ensure stem cell activation preferably in that hemithorax (Figure 1E) (Chaturvedi et al., 2017).
Note: After the injury, there will be melanization which can be easily identified as a black spot.
Transfer flies into food vial for recovery and process them for immunostaining after 5-6 h of recovery at least (Figures 1D and 1E).
Figure 1. An image guide for inducing injury at Dorsal Longitudinal Muscle. A. Canton-S fly showing the site of injury marked by a white circle; B. A pin with holder for inducing injury, Scale bar = 1 cm. C. Schematic of longitudinal section of fly thorax. The arrow depicts the direction of pin inducing injury to DLM (Dorsal longitudinal muscles, depicted by six purple rectangles). Arrowhead shows the point of contact between a pin and muscle fiber. Green and Blue boxes represent other flight muscles in thorax that are left mostly uninjured. D. Thoracic flight muscles (DLM) showing the site of an injury indicated by a dotted circle (white). Whole mount of flight muscles labeled by phalloidin (green) with all nuclei labeled by TOPRO-3 (blue). Scale bar = 50 µm. E. Simplified scheme depicting unfused muscle stem cells associated with flight muscles.
Fly DLM dissection
Take one adult injured Drosophila and anesthetize.
Note: A semi-alternative method has been given as reference (Weitkunat and Schnorrer, 2014).
Put the anesthetized fly in a petri dish containing 1x PBS (pH 7.5) and use sufficient amount of 1x PBS to keep the animal submerged.
Hold the fly abdomen with forceps and cut the head, legs, wings, and abdomen with fine scissors under a stereo microscope. Keep only the thoracic part of the fly for rest of the procedure.
Note: To avoid accidental poking into muscles, operate thoracic tissue henceforth using leg stumps or halteres.
Immunohistochemistry and confocal microscopy
Submerge the whole thorax in 4% PFA in 1x PBS taken in a Petri dish for chemical fixation for 20 min (see Recipes).
Take a glass slide and stick double sided tape on the slide.
Place the thorax on the double-sided tape slide and orient the fly thorax in such a way that the ventral side is up. Then stick it on the tape.
Make a sagittal cut with a fine razor blade/scissor by following the ventral midline and submerge the hemi-thoraces in 1x PBS solution by holding wings or legs using forceps.
Note: Try to use finely sharpened new blades for better sectioning.
Wash the hemi-thoraces by submerging in permeabilization solution.
Essentially perform the immunostaining and microscopy as mentioned in Fernandes et al., 1991 and Chaturvedi et al., 2017.
For staining F-actin, Phalloidin has been used at 1:500 dilution in 1x PBS.
For staining nuclei, TOPRO-3-Iodide has been used at 1:1,000 dilution in 1x PBS.
Limitations of the method and expertise required
If a pin fails to poke through cuticle – The pin has lost its sharpness and replace it with a new pin. A new pin should be used for every set of 20-30 flies for consistent results.
Diameter of injured area differs between flies – Try a new pin.
Death of Drosophila post injury – Avoid extensive injury to trachea on the ventral side of the fly.
Infection/yeast growth in the injured area – Sterilize the pin and holder using 70% ethanol.
Staining is too faint – Remove excess of tissue such as abdomen, head, and legs.
Over-fixation of samples can result in absent or low staining. To obtain better results, samples should be fixed only for 20 min using 4% paraformaldehyde (always freshly prepared from 16% stock using 1x PBS).
Muscle shows the absence of F-actin staining – Muscle injury can lead to areas of low or no phalloidin staining. Alternatively, anti-myosin staining can be used to observe the overall muscle structure.
The preferred way to stain all the nuclei is to use DAPI/Hoechst along with secondary antibody rather than mounting media with DAPI in it.
Data analysis
Image analysis and data processing were essentially performed as mentioned in Chaturvedi et al., 2017.
Recipes
70% (v/v) ethanol in ddH2O
1x Phosphate buffered saline (PBS) (Cold Spring Harbor Protocols)
NaCl 137 mM
KCl 2.7 mM
Na2HPO4 10 mM
KH2PO4 1.8 mM
Adjust pH to 7.5
4% paraformaldehyde for chemical fixation
Make 4% paraformaldehyde from 16% paraformaldehyde in 1x PBS
Permeabilization solution
PBS containing 0.3% Triton X-100
Blocking solution
PBS containing 0.3% Triton X-100 and 0.1% of bovine serum albumin (BSA)
Acknowledgments
This protocol was adapted from the article, Identification and functional characterization of muscle satellite cells in Drosophila by Chaturvedi et al., 2017. We thank National Centre for Biological Sciences, Tata Institute of Fundamental Research and the J C Bose Fellowship of the Government of India for funding. We acknowledge Central Imaging & Flow Cytometry Facility for using the confocal microscope and NCBS Fly facility. The authors are also thankful to Avishek Ghosh and Rajan Surendra Thakur from Prof. Raghu Padinjat’s laboratory for helping us to use the stereo microscope with a camera attachment. We have no conflict of interest to declare.
References
Chaturvedi, D., Reichert, H., Gunage, R. D. and VijayRaghavan, K. (2017). Identification and functional characterization of muscle satellite cells in Drosophila. Elife 6.
Fernandes, J., Bate, M. and Vijayraghavan, K. (1991). Development of the indirect flight muscles of Drosophila. Development 113(1): 67-77.
Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using μManager software. J Biol Methods 1(2).
Weitkunat, M. and Schnorrer, F. (2014). A guide to study Drosophila muscle biology. Methods 68(1): 2-14.
Copyright: Chakraborty et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Chakraborty, K., VijayRaghavan, K. and Gunage, R. D. (2018). A Method to Injure, Dissect and Image Indirect Flight Muscle of Drosophila. Bio-protocol 8(10): e2860. DOI: 10.21769/BioProtoc.2860.
Chaturvedi, D., Reichert, H., Gunage, R. D. and VijayRaghavan, K. (2017). Identification and functional characterization of muscle satellite cells in Drosophila. Elife 6.
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Category
Stem Cell > Adult stem cell > Muscle stem cell
Cell Biology > Tissue analysis > Tissue staining
Cell Biology > Tissue analysis > Tissue imaging
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2,861 | https://bio-protocol.org/exchange/protocoldetail?id=2861&type=0 | # Bio-Protocol Content
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Induction of Photothrombotic Stroke in the Sensorimotor Cortex of Rats and Preparation of Tissue for Analysis of Stroke Volume and Topographical Cortical Localization of Ischemic Infarct
Anna M. Wiersma
IW Ian R. Winship
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2861 Views: 11391
Edited by: Oneil G. Bhalala
Reviewed by: Miao HePatrick Ovando-Roche
Original Research Article:
The authors used this protocol in Nov 2017
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Nov 2017
Abstract
The photothrombotic model of stroke is commonly used in research as it allows the ischemic infarct to be targeted to specific regions of the cortex with high reproducibility and well-defined infarct borders. Unlike other models of stroke, photothrombosis allows the precise size and location of infarct to be tightly controlled with minimal surgical invasion. Photothrombosis is induced when a circulating photosensitive dye is irradiated in vivo, resulting in focal disruption of the endothelium, activation of platelets and occlusion of the microvasculature (Watson et al., 1985; Dietrich et al., 1987; Carmichael, 2005). The protocols here define how photothrombosis can be specifically targeted to the sensorimotor forelimb cortex of rat with high reproducibility. Detailed methods on rat cortical tissue processing to allow for accurate analysis of stroke volume and stereotactic determination of the precise cortical region of ischemic damage are provided.
Keywords: Photothrombosis Ischemic infarct Stroke model Stroke volume Ischemia
Background
The photothrombotic model of stroke allows for the precise placement of an ischemic infarct in specific regions of the cortex (Carmichael, 2005; Underly and Shih, 2017). Photothrombosis can be used to occlude specific arteries and arterial branches in the cortex (Carmichael et al., 2005), individual vessels of the pia (Taylor and Shih, 2013) and defined cortical areas such as the barrel field (Dietrich et al., 1987) and hind limb somatosensory cortex (Que et al., 1999). Using this approach, highly reproducible ischemic infarcts have been generated in many experimental animal models including rodents (Watson et al., 1985; Carmichael et al., 2005) and non-human primates (Ikeda et al., 2013). Photothrombosis of the forelimb sensorimotor cortex is useful as it results in localized sensorimotor impairment in forelimb use that can be carefully quantified using a variety of behavioral tests after stroke and during recovery (Sist et al., 2014; Wiersma et al., 2017). The methods presented here allows for the induction of consistent ischemic infarcts of the forelimb sensorimotor cortex that result in significant and long-lasting deficits in forelimb motor function (Wiersma et al., 2017). There is currently no standardized method of identifying both the volume of induced stroke and the anatomical location of stroke in the cortex. Here, we provide methods on systematic identification of the precise cortical location of photothrombotic infarct and analysis of stroke volume. Exclusion criteria for animal stroke models are widely varied. Therefore we provide guidelines to establish criteria for exclusion of animals that deviate from expected stroke volumes or stereotaxic locations.
Materials and Reagents
Surgical
Sterile scalpel blade # 10 (Fine Scientific Tools, catalog number: 10100-00 )
Blunt 16-gauge needle (STEMCELL Technologies, catalog number: 28110 )
1 ml syringes x 5 (BD, catalog number: 309659 )
I.V. catheter 24G x 5/8” (Smiths Medical, Jelco®, catalog number: 4073 )
Silk suture 5-0, P-3 reverse cutting (Ethicon, PERMAHAND®, catalog number: 640G )
Sprague-Dawley rat, male, ~500 g, 15-20 weeks of age (Charles River)
Isoflurane USP 99% (Fresenius Kabi, catalog number: CP0406V2 )
Compressed oxygen gas with high purity 99.995% (Praxair)
Compressed nitrous oxide gas 99% purity (Praxair)
BetadineTM surgical scrub (Fisher Scientific, catalog number: 19-027132)
Manufacturer: Purdue Pharma, catalog number: 6761815117 .
Sterile saline, 0.9% NaCl (Baxter, catalog number: 2B1324X )
Rose bengal (Sigma-Aldrich, catalog number: 330000 )
0.25% Bupivacaine (Sigma-Aldrich, catalog number: B5274 )
Buprenorphine (Schering- Plough, 0.2 mg injectable)
Rose bengal solution (see Recipes)
Perfusion
Euthasol® (VIRBAC, catalog number: 710101 )
Celline isotonic saline solution (Fisher Scientific, catalog number: 351142-10)
Manufacturer: SCP SCIENTIFIC, catalog number: CS20310D .
Heparin (Sigma-Aldrich, catalog number: H3393 )
Formalin 1:10 dilution, buffered (Fisher Scientific, catalog number: SF100-20 )
Isotonic saline heparin (5,000 IU/L) solution (see Recipes)
4% formalin solution (see Recipes)
Tissue Cryosectioning
Superfrost® plus gold slides (Fisher Scientific, catalog number: 22-035813 )
2-methylbutane (Sigma-Aldrich, catalog number: 277258 )
D-Sucrose (Fisher Scientific, catalog number: 10638403 )
Tissue Tek® OCT compound (Sakura, catalog number: 4583 )
30% sucrose solution (see Recipes)
Stroke Analysis
Rat atlas (Paxinos, George, and Charles Watson. The rat brain in stereotaxic coordinates: hard cover edition. Access Online via Elsevier, 2006. A tool by Matt Gaidica.)
Equipment
Heating pad with rectal thermometer (Stoelting, catalog number: 50300 )
Sterile Crile hemostat (Fine Science Tools, catalog number: 13004-14 )
Sterile blunt ended scissors (Fine Science Tools, catalog number: 14001-18 )
Sterile rongeur (Fine Science Tools, catalog number: 16000-14 )
Carbide burs drill bit (SS WHITE, catalog number: 14002-5 )
Perfusion tray (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 11469 )
Sterile scoop (Fine Science Tools, catalog number: 10090-13 )
Weigh scale (Kent Scientific, catalog number: SCL-1015 )
Electric razor, Mini cordless trimmer (Braintree Scientific, catalog number: CLP-9868 )
Animal anesthetic system (Harvard Apparatus, catalog number: 72-6467 )
Anesthetic induction chamber (Smiths Medical, catalog number: V711801 )
Stereotaxic frame (KOPF INSTRUMENTS, model: Model 963 Standard Accessories)
Surgical drill–TCM Endo III (Nouvag, catalog number: 31817 )
Laser 532 nm (Laserglow Technologies, model: LCS-0532 , Power Supply, Laserglow Technologies, model: C5310051X , with Thorlabs dielectric mirror to focus laser, Thorlabs, catalog number: CM1-E02 )
Laser safety goggles (Laserglow Technologies, catalog number: AGF5565XX )
Digital Peri-Star Peristaltic Pump (World Precision Instruments, catalog number: PERIPRO-4HS )
Cryostat (Leica, model: CM3050 S Research Cryostat)
Leica HCX PL APO L 10x 1.0NA water emersion objective lens
Leica SP5 Confocal microscope (Leica, model: Leica SP5 )
Animal surgery stereo microscope (Leica, M-series)
Inverted microscope (Leica, model: Leica DMI6000B )
Software
Leica LAS AF Software
ImageJ
Prism by Graph Pad vs9
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Wiersma, A. M. and Winship, I. R. (2018). Induction of Photothrombotic Stroke in the Sensorimotor Cortex of Rats and Preparation of Tissue for Analysis of Stroke Volume and Topographical Cortical Localization of Ischemic Infarct. Bio-protocol 8(10): e2861. DOI: 10.21769/BioProtoc.2861.
Wiersma, A. M., Fouad, K. and Winship, I. R. (2017). Enhancing spinal plasticity amplifies the benefits of rehabilitative training and improves recovery from stroke. J Neurosci 37(45): 10983-10997.
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Category
Neuroscience > Nervous system disorders > Animal model
Neuroscience > Nervous system disorders > Stroke
Cell Biology > Tissue analysis > Injury model
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2,862 | https://bio-protocol.org/exchange/protocoldetail?id=2862&type=0 | # Bio-Protocol Content
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In vivo Use of Dextran-based Anterograde Cortical Tracers to Assess the Integrity of the Cortical Spinal Tract
Anna M. Wiersma
IW Ian R. Winship
Published: Vol 8, Iss 10, May 20, 2018
DOI: 10.21769/BioProtoc.2862 Views: 6322
Edited by: Oneil G. Bhalala
Reviewed by: Trevor Martin SmithEhsan Kheradpezhouh
Original Research Article:
The authors used this protocol in Nov 2017
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Nov 2017
Abstract
When injected into the motor cortex of rats, anterograde tracers label fibers of the associated descending corticospinal tract (CST) that originate from pyramidal neurons in the tracer-injected cortex. These fibers can be assessed at the level of the spinal cord to determine the integrity of the descending CST and the spatial distribution of axons in the spinal grey matter. Here we provide detailed methods on the minimally invasive stereotaxic injection of anterograde tracers into the forelimb sensorimotor representation in the rat cortex. In addition, we detail the fixing and processing of spinal tissue for assessment of CST integrity and branching into spinal grey matter.
Keywords: Cortical spinal tract Anterograde tracer Spinal cord Animal model
Background
The motor cortex contains upper motor neurons which give rise to descending axons that form the CST and terminate in the spinal cord. Anterograde tracers injected into the motor cortex of rats label these descending fibers of the CST allowing their terminals to be assessed at the level of the spinal cord (Figure 1). In rodent models, anterograde labeling of the CST is widely used to assess CST integrity in a range of spinal cord injury models including lesions (Thallmair et al., 1998; Vavrek et al., 2006) and contusions (Hill et al., 2001). Further, anterograde cortical tracers have been used to assess spared fibers and plasticity of the CST in various models of brain injury such as cortical stroke (Wahl et al., 2014; Wiersma et al., 2017), traumatic brain injury (Ueno et al., 2012) and pyramidotomy (Starkey et al., 2012). Here we provide detail methods on injection of anterograde tracers into motor cortex corresponding the forelimb. We describe how to create burr holes without inducing damage to the cortex and the use of pulled glass pipette tips for minimally invasive injection. Since the CST is widely assessed in both brain and spinal cord injury models, we offer a systematic method of assessing the CST and branching fibers in the surrounding spinal grey matter that accounts for variability due to inter-animal differences in tracer efficacy or uptake.
Figure 1. Use of anterograde cortical tracers to label the corticospinal tract in rats. A. Tracer injection sites in the rat cortex (shown as green circle) and anterograde tracer labeled descending fibers (green line). B. Coronal sections of the brain at tracer injection sites. C. Transverse slices of the spinal cord (C4) with tracer labeled fibers of the CST shown in green.
Materials and Reagents
Surgical
Sterile scalpel blade # 10 (Fine Scientific Tools, catalog number: 10100-00 )
10 µl Hamilton syringe (Hamilton, catalog number: 80366 )
Glass microtubule–Glass filament with 1.0 mm external diameter (Sutter Instrument, catalog number: BF150-86-10 )
16-gauge needle (STEMCELL Technologies, catalog number: 28110 )
1 ml syringes x 5 (BD, catalog number: 309659 )
Silk suture 5-0, P-3 reverse cutting (Ethicon, PERMAHAND®, catalog number: 640G )
Sprague Dawley Rat, male, ~500 g,15-20 weeks of age (Charles River)
Isoflurane USP 99% (Fresenius Kabi, catalog number: CP0406V2 )
Compressed oxygen gas high purity 99.995% (Praxair)
Compressed nitrous oxide gas 99% purity (Praxair)
Sterile saline, 0.9% NaCl (Baxter, catalog number: 2B1324X )
0.25% Bupivacaine (Sigma-Aldrich, catalog number: B5274 )
Buprenorphine (Schering- Plough, 0.2 mg injectable)
Tracer
Dextran, Alexa FluorTM 488, 10,000 MW, Anionic, Fixable (Thermo Fisher Scientific, catalog number: D22910 )
Dextran amine, Texas Red®, 10,000 MW (Vector Laboratories, catalog number: SP-1140 )
Perfusion
Euthasol® (VIRBAC, catalog number: 710101 )
Celline isotonic saline solution (Fisher Scientific, catalog number: 351142-10)
Manufacturer: SCP SCIENTIFIC, catalog number: CS20310D .
Heparin (Sigma-Aldrich, catalog number: H3393 )
Formalin 1:10 dilution, buffered (Fisher Scientific, catalog number: SF100-20 )
4% formalin solution (see Recipes)
Isotonic saline heparin (5,000 IU/L) solution (see Recipes)
Tissue cryosectioning
Superfrost® Plus Gold slides (Fisher Scientific, catalog number: 22-035813 )
Isopentane (2-methylbutane) C5H12 (Sigma-Aldrich, catalog number: 277258 )
D-Sucrose (Fisher Scientific, catalog number: 10638403 )
Tissue Tek® Optimal cutting temperature (OCT) compound (Sakura, catalog number: 4583 )
VECTASHIELD (Vector Laboratories, catalog number: H-1000 )
30% Sucrose solution (see Recipes)
Spinal cord analysis
Rat atlas (Paxinos, George, and Charles Watson. The rat brain in stereotaxic coordinates: hard cover edition. Access Online via Elsevier, 2006. A tool by Matt Gaidica.)
Equipment
Heating pad (Stoelting, catalog number: 50300 )
Betadine® surgical scrub–(Fisher Scientific, catalog number: 19-027132 )
Sterile Crile Hemostats (Fine Science Tools, catalog number: 13004-14 )
Sterile blunt ended scissors (Fine Science Tools, catalog number: 14001-18 )
Sterile rongeur (Fine Science Tools, catalog number: 16000-14 )
Carbide burs drill bit (SS WHITE, catalog number: 14002-5 )
Perfusion tray (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 11469 )
Sterile scoop (Fine Science Tools, catalog number: 10090-13 )
Weigh scale (Kent Scientific, catalog number: SCL-1015 )
Animal Anesthetic System (Harvard Apparatus, catalog number: 72-6467 )
Stereotaxic frame (KOPF INSTRUMENTS, model: Model 963 with standard accessories)
Micropipette puller (Sutter Instrument, catalog number: P-30 )
Surgical drill–TCM Endo III (Nouvag, catalog number: 31817 )
Digital Peri-Star Pro-Peristaltic Pump (World precision instruments, catalog number: PERIPRO-4HS )
Cryostat (Leica Biosystems, catalog number: CM3050 S research cryostat)
Leica HCX PL APO L 10x 1.0NA water emersion objective lens
Leica SP5 Confocal microscope (Leica Microsystems, model: Leica SP5 )
Inverted microscope (Leica Microsystems, catalog number: Leica DMI6000B )
Animal surgery stereo microscope (Leica, M-series)
Software
Leica LAS AF Software
ImageJ
Prism by Graph Pad vs9
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Wiersma, A. M. and Winship, I. R. (2018). In vivo Use of Dextran-based Anterograde Cortical Tracers to Assess the Integrity of the Cortical Spinal Tract. Bio-protocol 8(10): e2862. DOI: 10.21769/BioProtoc.2862.
Wiersma, A. M., Fouad, K. and Winship, I. R. (2017). Enhancing spinal plasticity amplifies the benefits of rehabilitative training and improves recovery from stroke. J Neurosci 37(45): 10983-10997.
Download Citation in RIS Format
Category
Neuroscience > Nervous system disorders > Animal model
Neuroscience > Neuroanatomy and circuitry > Animal model
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2,863 | https://bio-protocol.org/exchange/protocoldetail?id=2863&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings
AP Agnès Pinel-Galzi
EH Eugénie Hébrard
OT Oumar Traoré
DS Drissa Silué
LA Laurence Albar
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2863 Views: 6829
Edited by: Feng Li
Reviewed by: Jinping ZhaoShankar Pant
Original Research Article:
The authors used this protocol in Dec 2013
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Dec 2013
Abstract
Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation – one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).
Keywords: Rice RYMV Inoculation Resistance ELISA Symptoms
Background
RYMV is a major biotic constraint for rice production in Africa (Séré et al., 2013) and has been reported in most rice-growing countries in Africa and Madagascar. It is not transmissible through seed (Konaté et al., 2001; Allarangaye et al., 2006) but by insect vectors (particularly beetles) and by contact during agricultural operations (Bakker, 1974; Traoré et al., 2006), especially while transplanting seedlings from seedbeds to the field. The virus is highly stable and capable of multiplying at high concentrations in its rice and wild Poaceae (Bakker, 1974) hosts.
Monitoring of RYMV incidence in seedbeds and varietal selection are the most efficient and sustainable ways of managing RYMV. There are two phenotypes of resistance – partial resistance, characterized by a delay in the appearance of symptoms (Albar et al., 1998), and high resistance, characterized by the absence of virus detection using DAS-ELISA (Ndjiondjop et al., 1999). Although partial resistance is widely distributed among Oryza sativa japonica varieties, only a few varieties from the cultivated rice species O. sativa and O. glaberrima express a high level of resistance to RYMV. Three major resistance genes – RYMV1, RYMV2 and RYMV3 – have been reported (Ndjiondjop et al., 1999; Thiémélé et al., 2010; Pidon et al., 2017).
Evaluation of the level of resistance to RYMV in rice varieties or lines and the comparison of the outcomes of different experiments require the use of a standardized protocol. This paper describes such a protocol which is based on DAS-ELISA and symptom severity. Reference accessions (susceptible, partially and highly resistant) have been included in the protocol to enable the drawing of reliable conclusions by comparing the test entry with reference materials. The number of plants tested for each variety or line is, however, not specified as it depends on the genetic material being tested and the objective of the experiment.
Materials and Reagents
Fontainebleau sand (VWR, catalog number: VWRC27460.295 )
Latex gloves
2 ml microcentrifuge tubes (Dominique DUTSCHER, catalog number: 033297 )
5 mm-steel beads (Brammer)
500 ml-wash bottle
Plates 96 wells (VWR, catalog number: 735-0083 )
10, 20, 200, and 1,000 μl tips
Paper towel
Rice seeds, including control accessions (susceptible, partially and highly resistant)
RYMV-infected rice leaves (fresh, dried or frozen at -20 °C)
Carborundum 0.037 mm used as an abrasive (VWR, catalog number: 22540.298 )
Liquid nitrogen (if possible)
RYMV antibody (wide spectrum polyclonal antibody against purified RYMV virions – prepared as described in N’Guessan et al. (2000) and Afolabi et al. (2009), or DSMZ, catalog number: AS-0732 or RT-0732 )
Alkaline phosphatase-conjugated polyclonal antibody against RYMV (prepared as described in Clark and Adams (1977), or DSMZ, catalog number: RT-0732 )
Skimmed milk powder
Substrate pNPP (para NitroPhenylPhosphate, Sigma-Aldrich, catalog number: N9389 )
Polyvinylpyrrolidone (PVP-40), MW 40000 (VWR, catalog number: 26616.184 )
Potassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
MilliQ water
Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
Sodium hydrogen carbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Tween 20 (Sigma-Aldrich, catalog number: P1379 )
Diethanolamine (Fisher Scientific, catalog number: 10131470 )
Hydrochloric acid (HCl) 10 N
Phosphate inoculation buffer (0.1 M pH 7.2) (see Recipes)
Buffers for ELISA test (see Recipes)
Buffer PBST 10x
Coating buffer pH 9.6
Substrate buffer pH 9.8 (see Recipes)
Equipment
Greenhouse or plant growth chamber
Mortars and pestles
Tissue lyser (QIAGEN, model: TissueLyser II )
10, 20, 200, and 1,000 μl volume pipettes
200 μl volume 8-channel pipette
Vortex
pH meter
Microcentrifuge
Microplate reader with 405 nm filter (TECAN, model: Infinite M200 Pro )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pinel-Galzi, A., Hébrard, E., Traoré, O., Silué, D. and Albar, L. (2018). Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings. Bio-protocol 8(11): e2863. DOI: 10.21769/BioProtoc.2863.
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Category
Plant Science > Plant immunity > Disease bioassay
Plant Science > Plant immunity > Host-microbe interactions
Biochemistry > Protein > Immunodetection
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2,864 | https://bio-protocol.org/exchange/protocoldetail?id=2864&type=1 | # Bio-Protocol Content
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Peer-reviewed
Root Gall Formation, Resting Spore Isolation and High Molecular Weight DNA Extraction of Plasmodiophora brassicae
Sara Mehrabi*
SS Suzana Stjelja*
CD Christina Dixelius
*Contributed equally to this work
Published: Jun 5, 2018
DOI: 10.21769/BioProtoc.2864 Views: 6253
Edited by: Samik Bhattacharya
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Abstract
Isolation of DNA from obligate biotrophic soil-borne plant pathogens is challenging. This is because of their strict requirement of living plant tissue for their growth and propagation. A soil habitat further imposes risk of contamination from other microorganisms living in close vicinity of the plant roots. Here we present a protocol on how to prepare DNA suitable for advanced molecular analysis on the soil-borne pathogen Plasmodiophora brassicae, a peculiar unicellular plant pathogenic organism, causing disease on Crucifers. First, it is important to grow Brassica or Arabidopsis plants in infested soils below a temperature of 25 °C under moist conditions to promote root gall formation. Root galls should be harvested ahead of initiation of the decomposing process, no later than four or nine weeks post inoculation of Arabidopsis or Brassica plants, respectively. Resting spores with reduced numbers of soil organisms are achieved by gradient centrifugations of homogenized gall tissues. Treatments with 70% alcohol and a suit of different antibiotics promote P. brassicae purity. A CTAB-based procedure allows isolation of high quality DNA suitable for massive parallel sequencing analysis.
Keywords: Arabidopsis Brassica Clubroot DNA Plasmodiophora brassicae Resting spores Rhizaria
Background
Plasmodiophora brassicae is a soil-borne plant pathogen causing root galls (clubs) in the Brassicaceae family including Arabidopsis. The clubroot disease has a major impact on oilseed rape (canola) and cabbage cultivation worldwide. P. brassicae is an obligate biotroph (require a host for growth) assigned to the supergroup Rhizaria, one of the least studied organism groups of eukaryotes (Sierra et al., 2016; Sibbald and Archibald, 2017). Phylogenetically, P. brassicae belongs to a plant pathogenic group of protists in Phytomyxea (Neuhauser et al., 2011 and 2014; Adl et al., 2012). Few genomes of related species are available, a circumstance which has considerably delayed the molecular analysis and genome comparisons. P. brassicae forms hardy resting spores in the clubs, spores that have the capacity to remain dormant for decades in the soil, ready for new rounds of root infections if a host plant grow nearby. Here we describe how to generate diseased plants, isolate resting spores from root galls followed by extraction of large amounts of DNA. This protocol is a further improvement and clarification of the procedures described in Schwelm et al. (2015). The outlined work is substantial but yields high-quality DNA suitable for long-read massive parallel sequencing.
Materials and Reagents
Materials
Safety glasses, gloves and lab coat
Filter paper
Plant pots, small pots (6 x 6 x 5 cm) and big pots (13 x 13 x 13 cm)
Plant trays (34 x 22 x 4 cm)
Soil (S-soil, Hasselfors Garden, Örebro, pH 5.5-6.5) composed of sighted light peat, black peat, perlite, sand, and lime
Petri dishes 10 cm (ø)
Miracloth Calbiochem® (Merck, catalog number: 475855 )
Tubes (Falcon tube 15 ml, SARSTEDT, catalog number: 62.554.001 ; Falcon tube 50 ml, SARSTEDT, catalog number: 62.547.004 ; Eppendorf micro-tube 2 ml, SARSTEDT, catalog number: 72.695.500 )
Plastic and glass beakers
Scalpel
Mortar, pestle and spoon (sterile and pre-chilled)
Filtropur S0.2 (SARSTEDT, catalog number: 83.1826.001 )
Plants
Brassica rapa cv. ‘Granaat’ (European Clubroot Differential Set ECD-05)
Arabidopsis thaliana Col-0
Plasmodiophorid
Plasmodiophora brassicae strain e3 (Fähling et al., 2004; the strain is available upon request to the authors)
Note: Not all strains incite disease on Arabidopsis.
Molecular biology working kit
SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, catalog number: STRN50 )
Other reagents
Note: *Those solutions are prepared with sterile distilled water.
Spore isolation
Ethanol (70%, 500 ml*)
Sodium hypochlorite (1%, 500 ml*) (Commercial bleach)
Sterile distilled water 5 x 1L
Ficoll PM 400 (16% and 32%*) (Sigma-Aldrich, catalog number: F4375 )
Rifampicin (100 mg/ml stock, prepared with methanol solvent) (Duchefa Biochemie, catalog number: R0146 )
Streptomycin sulfate (100 mg/ml stock*) (Thermo Fisher Scientific, catalog number: 11860038 )
Carbenicillin disodium (100 mg/ml stock, prepared with water solvent) (Duchefa Biochemie, catalog number: C0109 )
Pimaricin (100 mg/ml stock*) (Merck, Sigma-Aldrich, catalog number: 1.07360 )
Cefotaxime sodium (100 mg/ml stock*) (Duchefa Biochemie, catalog number: C0111 )
Hygromycin B (50 mg/ml stock) (Duchefa Biochemie, catalog number: H0192 )
Lysozyme from chicken egg white (4 mg/ml*) (Sigma-Aldrich, catalog number: L6876 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
Potassium chloride (KCl) (Merck, catalog number: 104936 )
di-Sodium hydrogen phosphate dihydrate (Na2HPO4) (Merck, catalog number: 119753 )
Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 104873 )
Tris-HCl (Trizma® hydrochloride solution) (1 M, pH 7.5) (Sigma-Aldrich, catalog number: T2694 )
DNase I, RNase-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0521 )
Proteinase K (20 mg/ml stock*) (Sigma-Aldrich, catalog number: RPROTK-RO )
EDTA (0.5 M) (VWR, catalog number: 20294.294 )
N-lauroylsarcosine sodium salt solution (1%, v/v) (Sigma-Aldrich, catalog number: 61747 )
1x PBS buffer (see Recipes)
1x TE buffer (pH 7.5) (see Recipes)
Termination buffer (see Recipes)
DNA extraction
Liquid nitrogen
Tris-HCl (Trizma® hydrochloride solution) (1 M, pH 7.5) (Sigma-Aldrich, catalog number: T2694 )
EDTA (VWR, catalog number: BDH9232 )
Sodium chloride (NaCl) (5 M stock *) (Sigma-Aldrich, catalog number: 31434 )
Hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H6269 )
2-Mercaptoethanol (VWR, catalog number: 436022A )
Phenol:chloroform:isoamyl alcohol 25:24:1 (pH 7.5-8.0) (Carl Roth, catalog number: A156.2 )
RNAse A, DNase and protease-free (10 mg/ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0531 )
Chloroform, EMSURE® ACS, ISO, Reag. Ph. (Merck, catalog number: 1.02445.1000 )
Ethanol (95%)
Note: Pre-chill at -20 °C before use.
Ethanol (70%)*
0.1x TE buffer (see Recipes)
CTAB extraction buffer (see Recipes)
Media and buffers (see Recipes)
1x PBS buffer
1x TE buffer
Termination buffer
CTAB extraction buffer
Equipment
Growth chamber (Percival AR82L2/Split) or greenhouse
Analytic balance (Mettler Toledo, model: AE100 )
Household mixer (Rusta, catalog number: 90951442 , Max power 170 W)
Liquid nitrogen container
Water bath (JULABO, model: Julabo TW12, catalog number: 9550112 )
pH meter (Mettler Toledo, model: SevenCompact S220, catalog number: 30019028 )
Tabletop centrifuge for Eppendorf tubes (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM FrescoTM 17 , catalog number: 75002420)
Tabletop centrifuge for Falcon-tubes (Eppendorf, model: 5804/5804 R , catalog number: 5805000327)
NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA)
Microscope (Zeiss, Axioplan; camera: Leica Microsystems, model: Leica DFC295 )
Stereoscope (Lecia, model: MZ FL III )
Autoclave
Procedure
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Category
Plant Science > Plant physiology > Biotic stress
Microbiology > Microbe-host interactions > Fungus
Molecular Biology > DNA > DNA extraction
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2,865 | https://bio-protocol.org/exchange/protocoldetail?id=2865&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Hyaluronan Isolation from Mouse Mammary Gland
CT Cornelia Tolg
MC Mary Cowman
ET Eva A. Turley
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2865 Views: 4769
Edited by: Vivien Jane Coulson-Thomas
Reviewed by: tarsis ferreira
Original Research Article:
The authors used this protocol in Nov 2017
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The authors used this protocol in:
Nov 2017
Abstract
The glycosaminoglycan hyaluronan (HA) is a key component of the extracellular matrix. The molecular weight of HA is heterogeneous and can reach from several million to several hundred daltons. The effect of HA on cell behavior is size dependent; fragmented HA acts as a danger signal, stimulates cell migration and proliferation and is proinflammatory, native high molecular weight HA suppresses inflammation. Therefore, it is important to analyze HA size distribution when studying the role of HA in tissue homeostasis and pathology. This protocol describes isolation of HA from mouse mammary glands but can also be applied to other tissues. The quality of the isolated HA is sufficient to analyze size distribution by gel electrophoresis (Calabro et al., 2000).
Keywords: Hyaluronan Size distribution Mammary gland Extracellular matrix Ion exchange fractionation
Background
The glycosaminoglycan HA consists of N-acetyl glucosamine and beta glucuronic acid disaccharides and is a ubiquitous component of the extracellular matrix. High molecular weight HA is fragmented by enzymatic degradation and oxidation by reactive oxygen and nitrogen species. In healthy tissues, the majority of HA is of high molecular weight. Accumulation of fragmented HA acts as danger signal during pathological processes (Tolg et al., 2012 and 2017; Yuan et al., 2015). For example, HA fragments stimulate inflammation whereas high molecular weight HA suppresses inflammation. HA influences cell behavior such as cell migration and proliferation by interaction with cell membrane receptors, leading to activation of signaling pathways. Since receptor-HA interactions are influenced by HA size, the effect of HA on tissue biology depends not only on HA amount but rather on HA size distribution and HA receptor expression by individual cells.
Materials and Reagents
Pipette tips (Fisher Scientific, catalog numbers: 02-707-417 , 02-707-408 , 02-707-438 )
1.5 ml Eppendorf microcentrifuge tubes (Diamed Advan Tech, catalog number: AD150-N )
Slide-A-Lyzer® MINI Dialysis Devices 7K MWCO, 0.1 ml (Thermo Fisher Scientific, catalog number: 69560 )
Slide-A-Lyzer® MINI Dialysis Devices 3.5K MWCO, 0.5 ml/2 ml (Thermo Fisher Scientific, catalog number: 88400 / 88403 )
Pierce Strong Anion Exchange Spin Column, Mini (Thermo Fisher Scientific, catalog number: 90010 )
C57Bl/6 female mice
Sterile de-ionized water
NaOH (BioShop, catalog number: SHY700.500 )
Tris base (121.1 g/mole) (BioShop, catalog number: TRS001.1 )
NaCl (58.44 g/mole) (BioShop, catalog number: SOD001.10 )
SDS (BioShop, catalog number: SDS001 )
CaCl2·2H2O (147.0 g/mole) (Sigma-Aldrich, catalog number: C3306 )
PBS (WISENT, catalog number: 311-425-CL )
Proteinase K (Roche Diagnostics, catalog number: 03115879001 ), recombinant, PCR grade
Note: It is approximately 2.5 U/mg when assayed with the Chromozym assay, or 30 U/mg with the hemoglobin assay.
Chloroform (reagent grade, Acros Organics, catalog number: 423555000 )
Ethanol (EtOH), 100%
Deferoxamine mesylate salt (=deferoxamine methanesulfonate salt) (657 g/mole) (Sigma Aldrich, catalog number: 1166003 )
Benzonase (Sigma-Aldrich, catalog number: E1014 ) recombinant 250 U/μl
0.2 M NaOH (see Recipes)
1 M Tris pH 8.3 (see Recipes)
5 M NaCl (see Recipes)
10% SDS (see Recipes)
1 M CaCl2 (see Recipes)
Proteinase K solution, 20 mg/ml (see Recipes)
Deferoxamine mesylate solution, 50 mg/ml (see Recipes)
Tissue lysis buffer (see Recipes)
0.1 M NaCl (see Recipes)
Benzonase digestion buffer (see Recipes)
NaCl solutions for ion exchange (IEX) fractionation (see Recipes)
Equipment
Pipettor (Drummond Scientific, Pipet-Aid®, model: XP2 )
Pipetman (Gilson, model: PIPETMAN® Classic )
Incubator capable to hold 37 °C (VWR, model: 1510 E )
Eppendorf Thermomixer (Eppendorf, model: ThermoMixer® R )
Refrigerated tabletop microcentrifuge capable of 18,000 x g (Thermo Fisher Scientific, model: SorvallTM LegendTM Micro 21R )
Rotor included with SorvallTM LegendTM Micro 21R
Flammable materials storage freezer, -20 °C
Fisher Scientific stirring Hotplate
Biohazard/Laminar flow hood
Digital Analytical Balance Scale (U.S. Solid Store, model: USS-DBS5 ); Repeatability: ± 0.2 mg
Balance scale (Denver Instrument, model: XS-310 D , Repeatability ± 0.02 g
pH meter (Fisher Scientific, model: accumetTM AR15 )
Fluorophore-assisted carbohydrate electrophoresis (FACE)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Tolg, C., Cowman, M. and Turley, E. A. (2018). Hyaluronan Isolation from Mouse Mammary Gland. Bio-protocol 8(11): e2865. DOI: 10.21769/BioProtoc.2865.
Download Citation in RIS Format
Category
Cancer Biology > General technique > Biochemical assays
Developmental Biology > Morphogenesis > Organogenesis
Cell Biology > Tissue analysis > Physiology
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2,866 | https://bio-protocol.org/exchange/protocoldetail?id=2866&type=1 | # Bio-Protocol Content
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Peer-reviewed
Total RNA Extraction from Dinoflagellate Symbiodinium Cells
TX Tingting Xiang
Published: Jun 5, 2018
DOI: 10.21769/BioProtoc.2866 Views: 4416
Edited by: Adam Idoine
Reviewed by: Manuel Aranda
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Abstract
Dinoflagellates are unicellular algae that can have photosynthetic or nonphotosynthetic lifestyles. Dinoflagellates in the genus Symbiodinium can enter endosymbiotic associations with corals, providing the metabolic basis for the highly productive and biologically diverse coral-reef ecosystems (Hoegh-Guldberg, 1999), as well as with other cnidarians, including sea anemones and jellyfish, and non-cnidarian hosts (Trench, 1993; Lobban et al., 2002; Mordret et al., 2016).
Here, I describe a protocol for isolating total RNA from Symbiodinium cells.
Keywords: Symbiodinium Coral Symbiosis RNA extraction DNA
Materials and Reagents
Gloves (E&K Scientific Products, catalog number: EK400-S )
Pipette tips 10/20 μl (USA Scientific, catalog number: 1120-3810 ), 200 μl (USA Scientific, catalog number: 1120-8810 ), 1,000 μl (USA Scientific, catalog number: 1126-7810 )
Microcentrifuge tube 1.5 ml (E&K Scientific Products, catalog number: 280150 )
Microcentrifuge tube 2.0 ml (Thermo Fisher Scientific, catalog number: 3463 )
Symbiodinium SSB01
Diethylpyrocarbonate (DEPC) treated water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9922 )
Liquid nitrogen
Glass beads, acid-washed (Sigma-Aldrich, catalog number: G8772 )
Chloroform:isoamyl alcohol (24:1, v/v, Sigma-Aldrich, catalog number: C0549 )
Phenol:chloroform:isoamyl alcohol (25:24:1, v/v, Sigma-Aldrich, catalog number: P2069 )
Ethanol (Absolute, PHARMCO-AAPER, catalog number: 111000200 )
8 M LiCl (Sigma-Aldrich, catalog number: L7026 )
RNase-Free DNase (QIAGEN, catalog number: 79254 )
Agarose (Thermo Fisher Scientific, Thermo Scientific TM, catalog number: 17850 )
Phenol solution (Sigma-Aldrich, catalog number: P4682 )
3 M sodium acetate (VWR, catalog number: 97062-812 )
Coral Pro Salt (Red Sea)
Tris 1 M solution, pH 9.0 (VWR, catalog number: 97062-940 )
Sodium chloride, NaCl (Sigma-Aldrich, catalog number: S9888 )
Tris (hydroxymethyl) aminomethane (Sigma-Aldrich, catalog number: 252859 )
Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: E9884 )
Sodium dodecyl sulfate, SDS (Sigma-Aldrich, catalog number: L3771 )
Artificial Sea Water (see Recipes)
RNA Lysis buffer (pH 9.0) (see Recipes)
RNA lysis buffer (pH 7.0) (see Recipes)
Equipment
Glass Erlenmeyer flask
RNase-Free glass bottle
Pipettes
0.2-2 μl (VWR, catalog number: 89079-960 )
2-20 μl (VWR, catalog number: 89079-964 )
20-200 μl (VWR, catalog number: 89079-970 )
100-1,000 μl (VWR, catalog number: 89079-974 )
5 ml (E&K Scientific Products, catalog number: EK-67044 )
Vortex (VWR, catalog number: 97043-562 )
Incubator
-20 °C freezer
Mini bead beater (Cole-Parmer, catalog number: EW-36270-07 )
Oven
Centrifuge (Sorvall, model: Sorvall RC 5C )
Electrophoresis instrument
Procedure
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Category
Microbiology > Microbe-host interactions > Algae
Molecular Biology > RNA > RNA extraction
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2,867 | https://bio-protocol.org/exchange/protocoldetail?id=2867&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Dual-probe RNA FRET-FISH in Yeast
Gable M. Wadsworth
RP Rasesh Y. Parikh
HK Harold D. Kim
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2867 Views: 7255
Edited by: Gal Haimovich
Reviewed by: Manuel D GaheteImre Gáspár
Original Research Article:
The authors used this protocol in Sep 2017
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Original research article
The authors used this protocol in:
Sep 2017
Abstract
mRNA Fluorescence In Situ Hybridization (FISH) is a technique commonly used to profile the distribution of transcripts in cells. When combined with the common single molecule technique Fluorescence Resonance Energy Transfer (FRET), FISH can also be used to profile the co-expression of nearby sequences in the transcript to measure processes such as alternate initiation or splicing variation of the transcript. Unlike in a conventional FISH method using multiple probes to target a single transcript, FRET is limited to the use of two probes labeled with matched dyes and requires the use of sensitized emission. Any widefield microscope capable of sensitive single molecule detection of Cy3 and Cy5 should be able to measure FRET in yeast cells. Alternatively, a FRET-FISH method can be used to unambiguously ascertain identity of the transcript without the use of a guide probe set used in other FISH techniques.
Keywords: RNA FISH Fluorescence In Situ Hybridization Saccharomyces cerevisiae Budding yeast Transcription Single molecule
Background
Quantification of the transcript distribution of single cells is typically accomplished by targeting mRNA with multiple probes to achieve a bright signal that can be distinguished from non-specifically bound probes (Raj and Tyagi, 2010). However, in some instances, there are features on the transcript such as splicing variants or alternative initiation sites that would be indistinguishable to a conventional FISH probe set. These isoform sequences can have short 50 nt uniquely identifying sequences. Using two probes, one can target either side of the junction with a FRET pair and quantify up to three classifications of mRNA isoform simultaneously, e.g., the isoform with both probes (FRET), the isoform with probe 1 only, and the isoform with probe 2 only. The reliance on a single fluorophore or pair of fluorophores requires sensitive detection through an EMCCD. Also, the detection efficiency of a probe for a sequence without other isoforms can be estimated using a FRET pair (Wadsworth et al., 2017).
Materials and Reagents
Pyrex bottle (Corning, PYREXTM, catalog number: 13951L )
Falcon tube 50 ml (VWR, catalog number: 89039-658 )
Falcon tube 15 ml (VWR, catalog number: 89039-666 )
Nitrile gloves (VWR, catalog number: 40101-348 )
Light-duty tissue wipers (VWR, catalog number: 82003-820 )
Lens cleaning tissues (Olympus, catalog number: C-0100 )
Aluminum foil
Pipette tips (VWR, catalog numbers: 89079-466 , 89079-460 , and 89079-472 )
Plastic cuvettes (BrandTech Scientific, catalog number: 759075D )
Culture flask (Corning, PYREXTM, catalog number: 4442-250 )
Microcentrifuge tube (Corning, Axygen®, catalog number: MCT-175-C )
Microcentrifuge tube rack (Thermo Fisher Scientific, catalog number: 5973-0015 )
Petri dish (VWR, catalog number: 25384-088 )
#1.5, 18 mm square coverslip (Fisher Scientific, catalog number: 12-518-108B )
Glass slide (Fisher Scientific, catalog number: 12-544-1 )
Saccharomyces cerevisiae strains (collaborators or ATCC)
Low Auto Fluorescence Immersion Oil (Thorlabs, catalog number: MOIL-30 )
Ethanol (VWR, catalog number: BDH1156 )
Methanol ≥ 99% ACS Spectrophotometric grade (Sigma-Aldrich, catalog number: 154903-2L )
RNase free water (Quality Biological, catalog number: 351-068-131 )
Flourophore labeled DNA oligo probes, HPLC purified (Integrated DNA technologies or Eurofins Scientific)
High Strength 5-min Epoxy (Amazon, B001QFGTHG)
Zymolyase-20T at 21 000 units/g (Zymolyase-20T, Seikagaku Business Corporation)
SD Complete (see Recipes)
Carbon, Nitrogen, and Salts (CNS)
Dextrose (Sigma-Aldrich, catalog number: G8270-25KG )
Ammonium sulfate (Sigma-Aldrich, catalog number: A4418-5KG )
Potassium phosphate monobasic (VWR, catalog number: MK710002 )
Magnesium sulfate (Sigma-Aldrich, catalog number: M2773-500G )
Sodium chloride (Fisher Scientific, catalog number: S671-500 )
Calcium chloride (Sigma-Aldrich, catalog number: C3306-250G )
Biotin (Sigma-Aldrich, catalog number: B4501-1G )
Calcium pantothenate (Sigma-Aldrich, catalog number: 21210-25G-F )
Vitamins and trace elements (Vitamix)
Folic acid (Fisher Scientific, catalog number: BP251910 )
Inositol (Sigma-Aldrich, catalog number: 57569-25G )
Niacin (Acros Organics, catalog number: 128291000 )
P-aminobenzoic acid (Sigma-Aldrich, catalog number: A9878-25G )
Pyridoxine HCl (Acros Organics, catalog number: 150770500 )
Riboflavin (Sigma-Aldrich, catalog number: R9504-25G )
Thiamine HCl (Sigma-Aldrich, catalog number: T4625-25G )
Boric acid (Sigma-Aldrich, catalog number: B6768-500G )
Copper sulfate (Sigma-Aldrich, catalog number: C1297-100G )
Potassium iodide (Avantor Performance Materials, catalog number: JT3168-4 )
Ferric chloride (Acros Organics, catalog number: 217091000 )
Manganese sulfate (Sigma-Aldrich, catalog number: M7634-100G )
Sodium molybdate 2 (Sigma-Aldrich, catalog number: 243655-5G )
Zinc sulfate (Sigma-Aldrich, catalog number: Z4750-100G )
Complete Supplement Mixture (CSM)
Adenine (Sigma-Aldrich, catalog number: A9126-25G )
Arginine (Sigma-Aldrich, catalog number: A5131-100G )
Aspartic acid (Acros Organics, catalog number: 105041000 )
Histidine (Sigma-Aldrich, catalog number: H8000-25G )
Isoleucine (Acros Organics, catalog number: 166170250 )
Leucine (Sigma-Aldrich, catalog number: L8000-100G )
Lysine (Sigma-Aldrich, catalog number: L5626-100G )
Methionine (Sigma-Aldrich, catalog number: M9625-25G )
Phenylalanine (Acros Organics, catalog number: 130311000 )
Threonine (Acros Organics, catalog number: 138930250 )
Tryptophan (Acros Organics, catalog number: 140590250 )
Tyrosine (Acros Organics, catalog number: 140641000 )
Uracil (Acros Organics, catalog number: 157300250 )
Valine (Acros Organics, catalog number: 140811000 )
Bacto-agar (BD, catalog number: 214030 )
Buffer B (see Recipes)
Sorbitol (Sigma-Aldrich, catalog number: S6021-1KG )
Potassium phosphate (dibasic) (Sigma-Aldrich, catalog number: P3786-500G )
Spheroplasting Buffer (see Recipes)
Vanadyl ribonucleoside complex (Fisher Scientific, catalog number: 50-812-650 )
Hybridization Buffer (see Recipes)
Dextran sulfate (Sigma-Aldrich, catalog number: D8906-10G )
Escherichia coli tRNA (Sigma-Aldrich, catalog number: R1753-500UN )
BSA (RNase free) (Fisher Scientific, catalog number: BP671-1 )
20x SSC (RNase free) (Thermo Fisher Scientific, catalog number: AM9763 )
Wash Buffer (see Recipes)
Formamide (RNase free) (VWR, catalog number: 97061-392 )
Imaging Buffer (see Recipes)
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich, catalog number: 238813-1G )
Tris-base (for making 200 mM, pH 8 Tris-HCl) (Fisher Scientific, catalog number: BP152-500 )
Protocatechuic acid (PCA) (Sigma-Aldrich, catalog number: 08992-50MG )
Protocatechuate-3,4-dioxygenase (PCD) (Sigma-Aldrich, catalog number: P8279-25UN )
Equipment
Pipettors (e.g., VWR, catalog number: 75786-304 )
Centrifuge (e.g., Thermo Fisher Scientific, model: SorvallTM LegendTM XTR , catalog number: 75004521)
Spectrophotometer (e.g., Eppendorf, catalog number: 2231000516 )
Micro-centrifuge (Eppendorf, catalog number: 022620100 )
Incubator (e.g., Thermo Fisher Scientific, catalog number: 50125590 )
Autoclave (e.g., YAMATO SCIENTIFIC, catalog number: SM300 )
TIRF/HILO widefield microscope capable of sensitized emission
Lenses (e.g., Thorlabs, catalog numbers: ACN127-020-A , LB1157-A)
Filters and dichroics (e.g., Semrock, catalog numbers: BLP01-635R-25 , FF650-Di01-25x36 , FF560/659-Di01-25x36 , FF01-593/40-25 )
Adjustable mechanical slit (Thorlabs, catalog number: VA100 )
Broadband mirrors (Thorlabs, catalog number: BB1-E02 )
Optical mounts, posts, post holders (Thorlabs)
Widefield microscope (e.g., Olympus, model: IX81 )
60x or 100x high NA objective (e.g., Olympus UPlanSApo 100X/1.4 Oil)
EMCCD camera (e.g., Andor Technology, model: iXonEM + )
Fiberport (Thorlabs, catalog number: PAF-X-11-PC-A )
Single mode fiberoptic cable (Thorlabs, catalog number: P5-460B-PCAPC-1 )
Laser illumination (e.g., solid state laser: Oxxius, model: LCX-532L-100 ; Coherent, catalog number: 1185055 )
Slide translation stage (e.g., Ludl Electronic Products, model: BioPoint2 X-Y Stage )
Software
Melting temperature calculator (IDT, http://www.idtdna.com/calc/analyzer)
Rna folding calculator (Mfold, http://unafold.rna.albany.edu/?q=mfold)
Sequence specificity check (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi)
Microscope control (Micromanager, https://micro-manager.org/)
Spot counting software (Fish-Quant, https://bitbucket.org/muellerflorian/fish_quant)
Matlab
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wadsworth, G. M., Parikh, R. Y. and Kim, H. D. (2018). Dual-probe RNA FRET-FISH in Yeast. Bio-protocol 8(11): e2867. DOI: 10.21769/BioProtoc.2867.
Download Citation in RIS Format
Category
Molecular Biology > RNA > RNA detection
Microbiology > Microbial genetics > Gene expression
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2,868 | https://bio-protocol.org/exchange/protocoldetail?id=2868&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Single-probe RNA FISH in Yeast
Gable M. Wadsworth
RP Rasesh Y. Parikh
HK Harold D. Kim
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2868 Views: 7371
Edited by: Gal Haimovich
Reviewed by: Chenchen Liu
Original Research Article:
The authors used this protocol in Sep 2017
Download PDF
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How to cite
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Cited by
Original research article
The authors used this protocol in:
Sep 2017
Abstract
Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique reduces the cost of performing FISH.
Keywords: RNA FISH Fluorescence In Situ Hybridization Saccharomyces cerevisiae Budding yeast Transcription Single molecule
Background
Precise quantification of the transcript profile of single cells is possible by single molecule Fluorescence In Situ Hybridization (smFISH). This procedure gives good signal to noise by targeting a single mRNA molecule with multiple fluorescently labeled DNA oligo probes (Raj and Tyagi, 2010). Using this scheme, mRNA of length shorter than 200 nucleotides cannot be detected. However, in most experiments, the absolute transcript copy number is less informative than the relative copy number. To detect short transcripts or sequences, a short single DNA oligo probe can be used. The detection efficiency of a single probe is greater than 50 percent when using a single fluorophore to count mRNA (Wadsworth et al., 2017).
Materials and Reagents
Pyrex bottle (Corning, PYREXTM, catalog number: 13951L )
Falcon tube 50 ml (VWR, catalog number: 89039-658 )
Falcon tube 15 ml (VWR, catalog number: 89039-666 )
Nitrile gloves (VWR, catalog number: 40101-348 )
Light-duty tissue wipers (VWR, catalog number: 82003-820 )
Lens cleaning tissues (Olympus, catalog number: C-0100 )
Aluminum foil
Pipette tips (VWR, catalog numbers: 89079-466 , 89079-460 , and 89079-472 )
Plastic cuvettes (BrandTech Scientific, catalog number: 759075D )
Culture flask (Corning, PYREXTM, catalog number: 4442-250 )
Microcentrifuge tube (Corning, Axygen®, catalog number: MCT-175-C )
Microcentrifuge tube rack (Thermo Fisher Scientific, catalog number: 5973-0015 )
Petri dish (VWR, catalog number: 25384-088 )
#1.5 18 mm square coverslip (Fisher Scientific, catalog number: 12-518-108B )
Glass slide (Fisher Scientific, catalog number: 12-544-1 )
Saccharomyces cerevisiae strains (collaborators or ATCC)
Low Auto Fluorescence Immersion Oil (Thorlabs, catalog number: MOIL-30 )
Ethanol (VWR, catalog number: BDH1156 )
Methanol ≥ 99% ACS Spectrophotometric grade (Sigma-Aldrich, catalog number: 154903-2L )
RNase free water (Quality Biological, catalog number: 351-068-131 )
Fluorophore labeled DNA oligo probes, HPLC purified (Integrated DNA technologies or Eurofins Scientific)
High Strength 5-min Epoxy (Amazon B001QFGTHG)
Zymolyase-20T at 21,000 units/g (Zymolyase-20T, Seikagaku Business Corporation)
SD Complete (see Recipes)
Carbon, Nitrogen, and Salts (CNS)
Dextrose (Sigma-Aldrich, catalog number: G8270-25KG )
Ammonium sulfate (Sigma-Aldrich, catalog number: A4418-5KG )
Potassium phosphate monobasic (VWR, catalog number: MK710002 )
Magnesium sulfate (Sigma-Aldrich, catalog number: M2773-500G )
Sodium chloride (Fisher Scientific, catalog number: S671-500 )
Calcium chloride (Sigma-Aldrich, catalog number: C3306-250G )
Biotin (Sigma-Aldrich, catalog number: B4501-1G )
Calcium pantothenate (Sigma-Aldrich, catalog number: 21210-25G-F )
Vitamins and trace elements (Vitamix)
Folic acid (Fisher Scientific, catalog number: BP251910 )
Inositol (Sigma-Aldrich, catalog number: 57569-25G )
Niacin (Acros Organics, catalog number: 128291000 )
P-aminobenzoic acid (Sigma-Aldrich, catalog number: A9878-25G )
Pyridoxine HCl (Acros Organics, catalog number: 150770500 )
Riboflavin (Sigma-Aldrich, catalog number: R9504-25G )
Thiamine HCl (Sigma-Aldrich, catalog number: T4625-25G )
Boric acid (Sigma-Aldrich, catalog number: B6768-500G )
Copper sulfate (Sigma-Aldrich, catalog number: C1297-100G )
Potassium iodide (Avantor Performance Materials, catalog number: JT3168-4 )
Ferric chloride (Acros Organics, catalog number: 217091000 )
Manganese sulfate (Sigma-Aldrich, catalog number: M7634-100G )
Sodium molybdate 2 (Sigma-Aldrich, catalog number: 243655-5G )
Zinc sulfate (Sigma-Aldrich, catalog number: Z4750-100G )
Complete Supplement Mixture (CSM)
Adenine (Sigma-Aldrich, catalog number: A9126-25G )
Arginine (Sigma-Aldrich, catalog number: A5131-100G )
Aspartic acid (Acros Organics, catalog number: 105041000 )
Histidine (Sigma-Aldrich, catalog number: H8000-25G )
Isoleucine (Acros Organics, catalog number: 166170250 )
Leucine (Sigma-Aldrich, catalog number: L8000-100G )
Lysine (Sigma-Aldrich, catalog number: L5626-100G )
Methionine (Sigma-Aldrich, catalog number: M9625-25G )
Phenylalanine (Acros Organics, catalog number: 130311000 )
Threonine (Acros Organics, catalog number: 138930250 )
Tryptophan (Acros Organics, catalog number: 140590250 )
Tyrosine (Acros Organics, catalog number: 140641000 )
Uracil (Acros Organics, catalog number: 157300250 )
Valine (Acros Organics, catalog number: 140811000 )
Bacto-agar (BD, catalog number: 214030 )
Buffer B (see Recipes)
Sorbitol (Sigma-Aldrich, catalog number: S6021-1KG )
Potassium phosphate (dibasic) (Sigma-Aldrich, catalog number: P3786-500G )
Spheroplasting Buffer (see Recipes)
Vanadyl ribonucleoside complex (Fisher Scientific, catalog number: 50-812-650 )
Hybridization Buffer (see Recipes)
Dextran sulfate (Sigma-Aldrich, catalog number: D8906-10G )
Escherichia coli tRNA (Sigma-Aldrich, catalog number: R1753-500UN )
BSA (RNase free) (Fisher Scientific, catalog number: BP671-1 )
20x SSC (RNase free) (Thermo Fisher Scientific, catalog number: AM9763 )
Wash Buffer (see Recipes)
Formamide (RNase free) (VWR, catalog number: 97061-392 )
Imaging Buffer (see Recipes)
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich, catalog number: 238813-1G )
Tris base (For making 200 mM, pH 8 Tris- HCl) (Fisher Scientific, catalog number: BP152-500 )
Protocatechuic acid (PCA) (Sigma-Aldrich, catalog number: 08992-50MG )
Protocatechuate-3,4-dioxygenase (PCD) (Sigma-Aldrich, catalog number: P8279-25UN )
Equipment
Pipettors (e.g., VWR, catalog number: 75786-304 )
x-y translation mount (Thorlabs, catalog number: ST1XY-S )
Fiberport (Thorlabs, catalog number: PAF-X-11-PC-A )
Note: This product has been superseded by part number PAF2P-11A .
Fiber optic cable (Thorlabs, catalog number: SM450 )
Single mode fiberoptic cable (Thorlabs, catalog number: P5-460B-PCAPC-1 )
Widefield microscope (e.g., Olympus, model: IX81 )
Spectrophotometer (e.g., Eppendorf, catalog number: 2231000516 )
Centrifuge (e.g., Thermo Fisher Scientific, model: SorvallTM LegendTM XTR , catalog number: 75004521)
Lenses (e.g., Thorlabs, catalog numbers: ACN127-020-A , LB1157-A)
Filters and dichroics (e.g., Semrock, catalog numbers: BLP01-635R-25 , FF650-Di01-25x36 )
Micro-centrifuge (Eppendorf, catalog number: 022620100 )
Incubator (e.g., Thermo Fisher Scientific, catalog number: 50125590 )
Autoclave (e.g., YAMATO SCIENTIFIC, catalog number: SM300 )
60x or 100x high NA objective (e.g., Olympus, UPlanSApo 100X/1.4 Oil)
EMCCD camera (e.g., Andor Technology, model: iXonEM + )
Laser illumination (e.g., solid state laser: Oxxius, model: LCX-532L-100 ; Coherent, catalog number: 1185055 )
Slide translation stage (e.g., Ludl Electronic Products, model: BioPoint2 X-Y Stage )
Software
Melting temperature calculator (IDT, http://www.idtdna.com/calc/analyzer)
RNA folding calculator (Mfold, http://unafold.rna.albany.edu/?q=mfold)
Sequence specificity check (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi)
Microscope control (Micromanager, https://micro-manager.org/)
Spot counting software (Fish-Quant, https://bitbucket.org/muellerflorian/fish_quant)
Matlab
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wadsworth, G. M., Parikh, R. Y. and Kim, H. D. (2018). Single-probe RNA FISH in Yeast. Bio-protocol 8(11): e2868. DOI: 10.21769/BioProtoc.2868.
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Category
Molecular Biology > RNA > RNA detection
Microbiology > Microbial genetics > Gene expression
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2,869 | https://bio-protocol.org/exchange/protocoldetail?id=2869&type=0 | # Bio-Protocol Content
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Peer-reviewed
Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa
MP Magdalena Pezzoni
RP Ramón A. Pizarro
Cristina S. Costa
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2869 Views: 8114
Edited by: Dennis Nürnberg
Reviewed by: Michael EnosDeena Jacob
Original Research Article:
The authors used this protocol in May 2016
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The authors used this protocol in:
May 2016
Abstract
Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental factors (e.g., ultraviolet radiation, osmotic stress), aerobic respiration, and host phagocytes during infective processes. Pseudomonas aeruginosa, a versatile bacterium, is a prevalent opportunistic human pathogen which possesses several defense strategies against ROS. Among them, two catalases (KatA and KatB) have been well characterized by their role on the defense against multiple types of stress. In this protocol, KatA and KatB activities are detected by polyacrylamide gel electrophoresis (PAGE). It is also suggested that the detection of KatB is elusive.
Keywords: Pseudomonas aeruginosa Catalase PAGE KatA KatB H2O2 Oxidative stress ROS
Background
P. aeruginosa is a ubiquitous bacterium that can be found in a free form in terrestrial and aquatics habitats or as an opportunistic human pathogen causing fatal infections in immunocompromised individuals, patients with skin damage or cystic fibrosis. To defend itself from ROS generated by its strong aerobic metabolism, host phagosomal vacuoles and environmental factors, this microorganism possesses several antioxidative strategies. Among them, two monofunctional catalases (KatA and KatB) are responsible for decomposing H2O2 to water and O2. KatA is the main catalase and has unique characteristics: it is unusually stable and essential to H2O2 resistance, osmoprotection and virulence (Hassett et al., 2000; Lee et al., 2005). It has been suggested that the stability of KatA is one of the main factors for the high level activity under normal growth conditions, and for this reason, katA has been regarded as a constitutively expressed gene in P. aeruginosa (Heo et al., 2010). However, it has been reported that KatA activity is induced in the stationary growth phase (up to 10-fold) and by increased levels of H2O2 (Brown et al., 1995; Suh et al., 1999; Heo et al., 2010). Moreover, katA expression has been demonstrated to be modulated by the global regulator OxyR and Quorum Sensing, whose activation depends on increased levels of H2O2 and high cell density, respectively (Hassett et al., 1999; Heo et al., 2010). KatB is only detected in the presence of H2O2 or paraquat and is partially involved in resistance to oxidative stress (Brown et al., 1995; Lee et al., 2005).
Solar ultraviolet-A (UVA) radiation is one of the main environmental stress factors for P. aeruginosa. Given the oxidative nature of UVA damage, we studied the role of catalases in defense of this microorganism against radiation. We demonstrated that KatA is essential in the optimal response against lethal doses of UVA, both in planktonic cells and biofilms (Costa et al., 2010; Pezzoni et al., 2014). In addition, we reported that low doses of UVA increase KatA and KatB activity and that this regulation occurs at the transcriptional level (Pezzoni et al., 2016). This phenomenon is relevant since it constitutes an adaptive mechanism that prevents cell damage by subsequent exposure to lethal doses of UVA, H2O2, or sodium hypochlorite (Pezzoni et al., 2016).
In the course of our studies, it became necessary to do an in-depth analysis of catalase activity. The total catalase activity in cell extracts was quantified by following spectrophotometrically the decomposition of H2O2, according to Aebi (1984). However, this assay cannot distinguish between KatA and KatB activities. To analyze individual catalase activity, we implement the method proposed by Wayne and Díaz (1986). In brief, crude cell extracts are loaded onto non-denaturing polyacrylamide gels (PAGE), and both catalases are separated by their differential electrophoretic motility; colorless bands of catalase activity are revealed by incubation of the gel with H2O2 and subsequent addition of a ferric chloride-potassium ferricyanide solution. The principle of this method involves the reaction of H2O2 with potassium ferricyanide (III) by reducing it to ferrocyanide (II); the peroxide is oxidized to O2. Ferric chloride reacts with ferrocyanide (II) to form an insoluble blue pigment. Because of the action of catalase on H2O2 decomposition, areas where this enzyme is active develop as clear bands in a blue gel (Patnaik et al., 2013). Additional papers were consulted to fine-tune this technique (Brown et al., 1995; Hassett et al., 1999; Elkins et al., 1999). The studies were performed with the prototypical P. aeruginosa strain PAO1 and isogenic derivatives PW8190 (katA::IslacZ/hah) and PW8769 (katB::IslacZ/hah) carrying mutations into katA and katB, respectively. Mutant strains devoid for KatA or KatB are useful to analyze the role of each enzyme in response to stress and as controls in PAGE catalase assays.
In this protocol, we describe how to detect individual catalase activity by PAGE using cell extracts from P. aeruginosa. Because of the particular characteristics of KatA (high abundance and stability), its detection does not present major difficulties. On the contrary, KatB detection is elusive, so that two changes were applied to the conventional technique: a protein extraction reagent was used instead of sonication to prepare the cell extracts, and the electrophoresis was performed at 4 °C. Based on these assays, it was concluded that KatB is an unstable enzyme, a fact that should be taken into account in quantitative or qualitative catalase assays under inducing (oxidative) conditions.
Materials and Reagents
Pipette tips
50 ml sterile conical Falcon tubes (Nunc® EZ FlipTM, Thermo Fisher Scientific, catalog number: 362696 )
1.5 ml sterile Eppendorf centrifuge tubes (Eppendorf, catalog number: 022364111 )
Sonication device
Note: This was assembled in our laboratory by attaching four plastic tubes (3 cm diameter, 3 cm high) to a plastic box (Figure 1).
Figure 1. Sonication device
Paper towel (WypAll* X 60 Jumbo Roll, KCWW, Kimberly-Clark, catalog number: 30218593 )
Spatula
Pyrex tray (Pyrex® Storage 13 x 18 cm)
P. aeruginosa strains
Note: PAO1, referred to as the wild-type, and catalase mutants PW8190 and PW8769 were obtained from the Washington Genome Center. Catalase mutants were constructed by insertion of IslacZ/hah transposon into katA (PW8190, hereinafter KatA-less strain) or katB (PW8769, hereinafter KatB-less strain) (Jacobs et al., 2003).
Distilled water
Tryptone (Oxoid, catalog number: LP0042 )
Yeast extract (Merck, catalog number: 103753 )
Sodium chloride (NaCl) (Biopack, catalog number: 1646.08 )
Albumin from bovine serum (Sigma-Aldrich, catalog number: A4378 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
Sodium phosphate (NaH2PO4) (Sigma-Aldrich, catalog number: S0751 )
Hydrogen peroxide (H2O2) 30% (Merck, catalog number: 107210 )
Bugbuster Protein Extraction Reagent (Merck, Novagen, catalog number: 70584-4 )
Sodium thiosulfate (Na2S2O3) (Avantor Performance Materials, MacronTM, catalog number: 8100-04 )
Ammonium persulfate ((NH4)2S2O8) (MP Biomedicals, catalog number: 04802811 )
TEMED (MP Biomedicals, catalog number: 02195516 )
Trizma base (Tris[hydroxymethyl]aminomethane) (C4H11NO3) (Sigma-Aldrich, catalog number: T1503 )
Glycine (Sigma-Aldrich, catalog number: G7126 )
Hydrochloric acid fuming 37% (HCl) (Merck, catalog number: 100317 )
Acrylamide (Sigma-Aldrich, catalog number: A8887 )
Bisacrylamide N,N’-methylene-bis-acrylamide (Sigma-Aldrich, catalog number: M7256 )
EDTA (Merck, Calbiochem, catalog number: 324503 )
Sodium hydroxide (NaOH) (Avantor Performance Materials, MacronTM, catalog number: 7708 )
Glycerol (Merck, catalog number: 104094 )
Bromophenol blue (VWR, DBH, catalog number: 20015 )
Ferric chloride (FeCl3) (Avantor Performance Materials, MacronTM, catalog number: 5029-04 )
Potassium ferricyanide (K3Fe (CN)6) (UCB, catalog number: b1599 )
LB medium (see Recipes)
4 M NaCl (see Recipes)
Saline solution (see Recipes)
50 mM sodium phosphate buffer, pH 7 (see Recipes)
30 mM H2O2 (see Recipes)
4 mM H2O2 (see Recipes)
10% ammonium persulfate (see Recipes)
1.5 M Tris-HCl buffer pH 8.8 (see Recipes)
1.5 M Tris-HCl buffer pH 6.8 (see Recipes)
30% acrylamide mix solution (acrylamide bisacrylamide ratio 37.5:1) (see Recipes)
6% resolving gel solution (see Recipes)
5% stacking gel solution (see Recipes)
1 M Tris-HCl buffer pH 8 (see Recipes)
0.5 M EDTA pH 8 (see Recipes)
Loading sample buffer (see Recipes)
Running buffer (see Recipes)
Ferric chloride/potassium ferricyanide solution (see Recipes)
Equipment
50, 125 and 150 ml sterile Erlenmeyer flasks (DWK Life Sciences, Duran®, catalog numbers: 21 216 17 , 21 216 28 , 21 990 27 )
2-20 µl, 20-100 µl, 100-1,000 µl Kartell pluripet micropipettes (Kartell LABWARE, catalog numbers: 13000 , 13210 , 13220 ) and 1-10 ml Acura® manual micropipette (Socorex, model: Acura® manual 825 / Acura® manual 835 )
50, 100 and 1,000 ml borosilicate measuring cylinders (VILABO, catalog number: 3501114 , 3501115 , 3501118 )
Conventional incubator shaker (New Brunswick Scientific, model: G25 )
Gyratory water bath shaker (New Brunswick Scientific, model: G76 )
Ice maker (Brema, model: TB 551 )
UV-Vis Spectrophotometer (Biotraza, model: 752 )
Refrigerated centrifuge (Hanil Scientific, model: Combi 514R )
Vibra-Cell sonicator (Sonics & Materials, model: VC500 )
Electrophoresis cell (Bioamerica, model: DYCZ-24DNBA )
Power supply (Bioamerica, model: DYY-6CBA )
Freezer ultra-low temperature (Sanyo, model: MDF-U76VC )
Autoclave (HIRAYAMA, HICLAVETM, model: HVE-50 )
Hot air oven sterilizer (Dalvo Intrumentos, model: OHR/T )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pezzoni, M., Pizarro, R. A. and Costa, C. S. (2018). Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa. Bio-protocol 8(11): e2869. DOI: 10.21769/BioProtoc.2869.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Activity
Biochemistry > Protein > Electrophoresis
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287 | https://bio-protocol.org/exchange/protocoldetail?id=287&type=0 | # Bio-Protocol Content
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In vitro RNA-protein Binding Assay by UV Crosslinking
Hailong Zhang
MZ Muxiang Zhou
Published: Vol 2, Iss 21, Nov 5, 2012
DOI: 10.21769/BioProtoc.287 Views: 25288
Original Research Article:
The authors used this protocol in Mar 2012
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Abstract
Because covalent bond can form between RNA and its binding proteins after UV irradiation, UV cross-linking is widely used to identify the specific RNA binding proteins. This protocol is described in details as follows.
Materials and Reagents
Linearized DNA template for transcription of the RNA of interest
MAXIscript in vitro Transcription Kit (Applied Biosciences, catalog number: AM1308M ) (This kit contain ATP, CTP, GTP, α-P32UTP and DNase I)
RNasin (Promega Corporation, catalog number: N2611 )
RNase T1 (Applied Biosciences, catalog number: 10109193001 )
0.5 M EDTA (Life Technologies, Gibco®, catalog number: 15575-038 )
ddH2O (GeneMate Loyalty, catalog number: UPW-1000 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
MgCl2 (Sigma-Aldrich, catalog number: M-2393 )
Glycerol (Sigma-Aldrich, catalog number: G7893-1L )
DTT (Sigma-Aldrich, catalog number: D9779-5G )
Tris-Base (Thermo Fisher Scientific, catalog number: BP152-1 )
SDS (Sigma-Aldrich, catalog number: L-4390 )
Bromphenol blue (Sigma-Aldrich, catalog number: B5525 )
TEMED
Ammoniampersulfate
5x binding buffer (see Recipes)
2x SDS loading buffer (see Recipes)
10x TGE buffer (see Recipes)
6% TGE gel (see Recipes)
Equipment
SPIN-PureTM column (G-50) (PireBiotech SCW50-50 DEPC-water)
SDS-PAGE system (Bio-Rad, catalog number: 165-1802 )
UV Stratalinker 1800 (Stratagene, catalog number: 474645 )
Film processor (Konica Minolta, model: SRX-101A )
Centrifuges (Eppendorf, catalog number: 5810R )
Scintillation Counter (Beckman, catalog number: LS6500 )
Bench top Radiation Shield with 6-1/4" base (Cole-Parmer, catalog number: WU-36218-00 )
37 °C water bath
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhang, H. and Zhou, M. (2012). In vitro RNA-protein Binding Assay by UV Crosslinking. Bio-protocol 2(21): e287. DOI: 10.21769/BioProtoc.287.
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Category
Biochemistry > RNA > RNA-protein interaction
Biochemistry > Protein > Interaction
Molecular Biology > RNA > RNA-protein interaction
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2,870 | https://bio-protocol.org/exchange/protocoldetail?id=2870&type=0 | # Bio-Protocol Content
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The Traveling Salesman Problem (TSP): A Spatial Navigation Task for Rats
RB R. E. Blaser
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2870 Views: 4602
Edited by: Jena Hales
Reviewed by: Beatriz Castro
Original Research Article:
The authors used this protocol in Nov 2015
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Nov 2015
Abstract
The Traveling Salesman Problem (TSP) is a behavioral test used to measure the efficiency of spatial navigation. It is an optimization problem, in which a number of baited targets are placed in an arena, and as the subject travels between the targets, the route is recorded and compared to chance and optimal routes. The TSP is appealing for the study of learning, memory, and executive function in nonhuman animals because the memory requirements can easily be modified with minor adjustments to task parameters. In the standard version of the task, rats are initially pre-trained to forage for bait in the arena. Once the animals consistently retrieve the bait, they are tested with a set of novel target configurations, and their behavior is recorded. The videos are then scored to produce several measures of performance.
Keywords: Spatial navigation Cognition Planning Working memory Executive function Optimization
Background
The Traveling Salesman Problem (TSP) is a spatial navigation task in which participants are asked to select the shortest possible route that visits each target in a spatial array. This task has been of interest to computer and cognitive scientists for some time (Best, 2006), but has more recently been studied from the perspective of comparative psychology (e.g., de Jong et al., 2011; Gibson et al., 2012; Baron et al., 2015). Humans are inexplicably good at finding solutions to this problem – despite its mathematical intractability, human participants can rapidly and easily produce solutions that are nearly optimal (MacGregor et al., 2000). One possible explanation for this high level of performance is that the task capitalizes on evolved processes used for natural behaviors like foraging (Blaser and Ginchansky, 2012; Blaser and Wilber, 2013). We were therefore interested in the mechanisms by which non-human animals complete the task (Bellizzi et al., 2015; Goldsteinholm and Blaser, 2015).
Although human participants typically use either a computer or paper and pencil to solve the TSP, we used the radial arm maze as a model to design a comparable task for rats (Blaser and Ginchansky, 2012). The task has not been widely used with nonhuman animals, but already several variant procedures exist. For example, Bures et al. (1992) used a TSP design in which animals were not rewarded until after all targets had been visited, with subjects trained on the same target configurations for 10 consecutive days prior to testing. Miyata et al. (2010) required pigeons to peck at a touch-screen in order to receive a food reward at the end of the test. Important variations across procedures involve whether food is available at each target or only at the end, whether animals are exposed to a specific spatial configuration more than once, and the scale of the spatial arena relative to the body size of the animal. We selected a protocol in which novel configurations are tested to reduce the degree to which performance relies on long-term spatial memory, and in which a relatively large arena encourages distance minimization.
Materials and Reagents
Rats
Ethanol
Targets
Semi-transparent purple vinyl index card dividers (Oxford Esselte, catalog number: 73153 )
Rodent food dishes
Sterilized bottle caps
Targets are pieces of plastic cut into 2” diameter circles. We use semi-transparent purple vinyl index card dividers by Oxford Esselte, but in the past have successfully used both rodent food dishes and sterilized bottle caps. The flat vinyl targets are appealing because they are simple to clean, and are less attractive to the rats to explore and carry, compared to dishes or caps. Whichever targets are employed, the targets used in a single experiment should be identical to each other.
Bait
Froot Loops® cereal (or a generic equivalent)
Fruity Pebbles® or Cocoa pebbles® (one pebble per target)
For bait, we use Froot Loops® cereal (or a generic equivalent) broken into quarter pieces (one quarter per target), or Fruity Pebbles® or Cocoa pebbles® (one pebble per target). All work equally well.
Configuration Templates
During pre-training, targets are placed randomly and do not require templates (see Procedure). In the test trials, however, the spatial arrangement of targets is critical. For this purpose, we create a template out of posterboard which is cut to fill the entire arena floor. Holes (approx. 2.5” diameter) are cut in the posterboard where the targets will be placed. Before each trial, the template is placed in the open field, and targets are then placed in the holes. The template can then be lifted out of the open field, leaving the targets located precisely in the arena.
Equipment
Open Field Arena (Figure 1)
The animals are tested in a 90 x 90 x 75 cm open field arena made of plywood. It is painted white, with a black grid painted on the floor. Any large standard open field should work, although the same type should be used consistently since the arena size and shape are expected to affect task performance measures.
Figure 1. Photo of the open field arena with ten baited targets and a rat
Stopwatch
A simple stopwatch (minutes/seconds) is used to time the animals in the task.
Video Camera
A video camera recording high-definition video files to SD card is used to record all trials. The video camera resolution must be sufficient to see (1) whether a piece of bait has been removed from the target in low lighting conditions, and (2) whether the vibrissa (whiskers) of the animal contact the target.
White Noise generator (e.g., HoMedics, model: SS-2000G/F-AMZ )
We use a standard home-use white noise generator, HoMedics SS-200G/F-AMZ. Any standard model of white-noise generator designed for home or laboratory use would be appropriate. The white noise generator helps to mask any distractions due to extraneous sounds in or near the testing room, which reduces behavioral variability in the task.
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Blaser, R. E. (2018). The Traveling Salesman Problem (TSP): A Spatial Navigation Task for Rats. Bio-protocol 8(11): e2870. DOI: 10.21769/BioProtoc.2870.
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Category
Neuroscience > Behavioral neuroscience > Learning and memory
Neuroscience > Behavioral neuroscience > Animal model
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2,871 | https://bio-protocol.org/exchange/protocoldetail?id=2871&type=0 | # Bio-Protocol Content
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Peer-reviewed
Intracellular and Mitochondrial Reactive Oxygen Species Measurement in Primary Cultured Neurons
SB Seung Hyun Baek
YC Yoonsuk Cho
JL Jeongmi Lee
BC Bo Youn Choi
YC Yuri Choi
JP Jin Su Park
HK Harkkyun Kim
JS Jaehoon Sul
EK Eunae Kim
JP Jae Hyung Park
DJ Dong-Gyu Jo
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2871 Views: 10117
Edited by: Xi Feng
Reviewed by: Welsch Charles Jeremy
Original Research Article:
The authors used this protocol in May 2017
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Abstract
Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules. ROS consist of radical oxygen species including superoxide anion (O2•−) and hydroxyl radical (•OH) and non-radical oxygen species such as hydrogen peroxide (H2O2), singlet oxygen (O2). ROS are generated by mitochondrial oxidative phosphorylation, environmental stresses including UV or heat exposure, and cellular responses to xenobiotics (Ray et al., 2012). Excessive ROS production over cellular antioxidant capacity induces oxidative stress which results in harmful effects such as cell and tissue damage. Sufficient evidence suggests that oxidative stresses are involved in cancers, cardiovascular disease, and neurodegenerative diseases including Alzheimer’s disease and Parkinson disease (Waris and Ahsan, 2006). Though excessive level of ROS triggers detrimental effects, ROS also have been implicated to regulate cellular processes. Since ROS function is context dependent, measurement of ROS level is important to understand cellular processes (Finkel, 2011). This protocol describes how to detect intracellular and mitochondrial ROS in live cells using popular chemical fluorescent dyes.
Keywords: Reactive oxygen species (ROS) Intracellular ROS MitoSOX CM-H2DCFDA Primary neuron
Background
ROS are important to maintain homeostasis in our bodies (Brieger et al., 2012). Many diseases such as cancer, neurodegenerative disease, cardiovascular disease, and diabetics are associated with ROS (Datta et al., 2000). DNA damage caused by ROS is a major cause of accelerating carcinogenesis process, and therapeutic agents targeting ROS have been actively developed (Trachootham et al., 2009). In circulatory system, abnormal oxidative stress increases the production of ROS, leading to various cardiovascular diseases (Forstermann, 2008). Signaling related to diabetes is sensitive to ROS, and these signaling abnormalities induced by abnormal levels ROS cause diabetes complications (Baek et al., 2017). Controlling the ROS levels in the brain is one of the most important activities because abnormal levels of ROS can cause diverse brain diseases. Amyloid beta, known as an important factor in Alzheimer’s disease, causes excessive ROS generation in the brain, neuronal damage (Singh et al., 2011), and eventually dementia (Polidori, 2004). Activated microglia produced by ROS which secretes a variety of cytokines result in neuronal death (Heneka et al., 2014).
ROS are generated by small part of oxygen consumed in mitochondria. A principal species of ROS produced in mitochondria is superoxide anion and it is the byproduct of the electron transport chain (Batandier et al., 2002). In order to detect superoxide in mitochondria, MitoSOX red, a mitochondria superoxide indicator, is used. Due to the positive charge on triphenylphosphonium group, MitoSOX red can effectively penetrate phospholipid bilayer, and accumulate into the matrix of mitochondria. Furthermore, hydroethidine of MitoSOX red allows researchers to discriminate the fluorescent signal generated by superoxide-mediated oxidative products from other non-specific signals (Robinson et al., 2006; Baek et al., 2017).
CM-H2DCFDA is a chloromethyl derivative of H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate), a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity within the cell and can be used to detect the intracellular formation of ROS (Kirkland et al., 2007). Once the fluorescent probe of CM-H2DCFDA permeates cell membrane, intracellular esterases hydrolyze its acetyl groups and it can be retained in the cell. CM-H2DCFDA is more sensitive to oxidation by H2O2 than superoxide (O2•−) (Fowler et al., 2017). CM-H2DCFDA is widely used in physiological and pathophysiological studies including virus infection (Nykky et al., 2014), cancer (Khatri et al., 2015; Liu et al., 2017), and neurodegenerative diseases (Ng et al., 2014). Using CM-H2DCFDA, we can detect intracellular ROS level by flow cytometry/fluorescence measurement and the localization of ROS producing organelle with confocal microscopy (Forkink et al., 2010).
Materials and Reagents
Glass bottom cell culture dish type 35 mm and dimension 20 mm (Nest Scientific, catalog number: 801001 )
Cover glasses thickness No. 1 circular size 18 mm Ø (MARIENFELD, catalog number: 0111580 )
Petri dish, 100 mm Polysterene aseptic non-tissue culture treated (SPL Life Sciences, catalog number: 10095 )
15 ml conical tube (SPL Life Sciences, catalog number: 50015 )
10 ml Serological pipettes (SPL Life Sciences, catalog number: 91010 )
50 ml conical tube (SPL Life Sciences, catalog number: 50050 )
Cell strainer 70 μm (Corning, Falcon®, catalog number: 352350 )
Pregnant female Sprague Dawley rats (E17-E18 days gestation, Orient Korea)
Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P6407-5mg )
Phosphate buffered saline powder, pH 7.4, for preparing 1 L solutions (Sigma-Aldrich, catalog number: P3813 )
CM-H2DCFDA (Thermo Fisher Scientific, InvitrogenTM, catalog number: C6827 )
Dimethyl Sulfoxide(DMSO) (Merck, catalog number: 317275 )
MitoSOXTM Red Mitochondrial Superoxide Indicator, for live-cell imaging (Thermo Fisher Scientific, InvitrogenTM, catalog number: M36008 )
Phosphate buffered saline (PBS) powder, pH 7.4, for preparing 1 L solutions, suitable for cell culture(Sigma-Aldrich, catalog number: P3813 )
Trypsin (2.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
Neurobasal Medium® (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
B-27TM Supplement (50x), serum free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
DMEM High Glucose (4.5 g/L), with L-Glutamine, with Sodium Pyruvate (Capricorn Scientific, catalog number: DMEM-HPA )
Penicillin/Streptomycin (100x) (PS) (Capricorn Scientific, catalog number: PS-B )
Amyloid beta peptide 1-42 Human (ANYGEN, catalog number: AGP-8338 )
CM-H2DCFDA solution (see Recipes)
MitoSOXTM Red solution (see Recipes)
Poly-D-lysine hydrobomide solution (see Recipes)
Prep medium (see Recipes)
Culture medium (see Recipes)
Maintain culture medium (see Recipes)
Equipment
Haemacytometers (MARIENFELD, catalog number: 0630010 )
Original Portable Pipet-Aid® Pipette Controller (Drummond Scientific, catalog number: 4-000-100 )
Dressing Scissors (Surgimax Instruments, catalog number: 85-112-12 ) (Figure 1A 1)
Dissecting Scissors (Surgimax Instruments, catalog number: 85-127-10 ) (Figure 1A 2)
Dissecting Scissors (Surgimax Instruments, catalog number: 63-175-11 ) (Figure 1A 3)
Spring Dressing Forceps Sharp (Surgimax Instruments, catalog number: 85-076-11 ) (Figure 1A 4)
Spring Dressing Forceps Blunt (Surgimax Instruments, catalog number: 85-073-15 ) (Figure 1A 5)
Multi Purpose Forceps Pointed (Surgimax Instruments, catalog number: 05-177-11 ) (Figure 1A 6)
Clean bench (HANBAEK Scientific Technology, catalog number: HB-402 )
Cell culture CO2 incubator (ARA, catalog number: APR150 )
Water-bath (Grant Instruments, JB Academy, catalog number: JBA18 )
Centrifuge (Hanil Scientific, catalog number: Combi 514R )
Confocal microscope with live cell imaging system (Carl Zeiss, model: LSM700 ) (Figures 1B and 1C)
Figure 1. Equipment for the experiment. A. Surgery instruments; B. Confocal microscope (LSM700) with live cell imaging system; C. Live cell chamber.
Software
For measure
ZEN black version (ZEISS confocal microscope LSM700 software)
Note: This is default program provided with ZEISS confocal microscope.
For analysis
ZEN Blue edition (ZEISS confocal microscope LSM700 software; SR-DIP software for ZEN blue edition)
ImageJ (ImageJ is an open source image processing program)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Baek, S. H., Cho, Y., Lee, J., Choi, B. Y., Choi, Y., Park, J. S., Kim, H., Sul, J., Kim, E., Park, J. H. and Jo, D. (2018). Intracellular and Mitochondrial Reactive Oxygen Species Measurement in Primary Cultured Neurons. Bio-protocol 8(11): e2871. DOI: 10.21769/BioProtoc.2871.
Baek, S. H., Park, S. J., Jeong, J. I., Kim, S. H., Han, J., Kyung, J. W., Baik, S. H., Choi, Y., Choi, B. Y., Park, J. S., Bahn, G., Shin, J. H., Jo, D. S., Lee, J. Y., Jang, C. G., Arumugam, T. V., Kim, J., Han, J. W., Koh, J. Y., Cho, D. H. and Jo, D. G. (2017). Inhibition of Drp1 ameliorates synaptic depression, Aβ deposition, and cognitive impairment in an Alzheimer's disease model. J Neurosci 37(20): 5099-5110.
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Category
Neuroscience > Cellular mechanisms > Intracellular signalling
Neuroscience > Nervous system disorders > Cellular mechanisms
Cell Biology > Cell imaging > Confocal microscopy
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2,872 | https://bio-protocol.org/exchange/protocoldetail?id=2872&type=0 | # Bio-Protocol Content
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Peer-reviewed
Slow and Fast Desiccation of Single-cell Thick Fronds of Filmy Ferns
MG Marcelo Garcés
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2872 Views: 4514
Original Research Article:
The authors used this protocol in Nov 2017
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Abstract
Filmy ferns can desiccate and recover after rehydration to resume photosynthesis. Slow and fast desiccation rates were compared in filmy fern fronds to determine whether structural or physiological differences may occur between desiccation rates. Slow desiccation is considered to be more similar to natural conditions experienced by plants that grow under the forest canopy. A fast desiccation rate will help to understand whether slow desiccation is important for recovery and viability.
Keywords: Desiccation tolerance Filmy fern Desiccation Hymenophyllaceae
Background
The Hymenophyllaceae is a family of epiphytic pteridophytes highly endemic to shady, constantly humid forest (Figure 1). There are some species of this filmy fern group that can survive desiccation to 20% relative water content, remain in this state for an extended period, and survive following rehydration (Figure 2) (Garcés et al., 2018) (See example timelapse, Video 1).
Figure 1. Hymenophyllum caudiculatum growing in nature under the forest canopy
Figure 2. Detached Hymenophyllum caudiculatum fronds under different conditions (Modified from Garcés, 2014). (Top) fresh detached, (middle) desiccated, and (bottom) rehydrated.
Video 1. Example of curling of Hymenophyllum caudiculatum fronds after desiccation/rehydration
Early studies of desiccation tolerance of Tortura ruralis examined slow or rapid dehydration on 500 mg fresh moss tissue (Dhindsa, 1987). Rapid desiccation was imposed by placing the tissue over activated silica gel granules in a desiccator (Relative Humidity [RH] of nearly 0%). Slow desiccation was administered by placing tissue samples over a stirred, saturated solution of ammonium nitrate contained in a desiccator (65% RH). A final weight of less than 20% original fresh weight was obtained in about 8 h of slow drying and in less than 30 min of rapid drying.
As ammonium nitrate is a hazardous and regulated substance in several countries, in this work, the slow desiccation solution was replaced by a saturated solution of potassium chloride (RH 75%).
Materials and Reagents
Absorbent paper
Petri dishes (Merck, catalog number: CORM3160-150X15 )
Silica gel 2-5 mm (Merck, catalog number: 1077351000 )
Note: Dry in an oven for 24 h at 60 °C.
Filmy fern fronds collected from Katalapi Park, Chile (Garcés et al., 2018).
Note: Fully expanded fronds of about 20 cm long were used in this protocol.
Potassium chloride (KCl) (Merck, catalog number: 1049361000 )
Saturated KCl solution (see Recipes)
Equipment
Glass desiccator, amber 300 mm (W.W. Grainger, model: 5YHV5 )
Analytical scale (Sartorius, catalog number: BP221S )
Forced air drying oven 50 L (Biobase, catalog number: BOV-T50F )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Garcés, M. (2018). Slow and Fast Desiccation of Single-cell Thick Fronds of Filmy Ferns. Bio-protocol 8(11): e2872. DOI: 10.21769/BioProtoc.2872.
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Category
Plant Science > Plant physiology > Abiotic stress
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Confocal microscopy
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2,873 | https://bio-protocol.org/exchange/protocoldetail?id=2873&type=0 | # Bio-Protocol Content
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Characterization of Protein Domain Function via in vitro DNA Shuffling
KP Kathy Hiu Laam Po
EC Edward Wai Chi Chan
SC Sheng Chen
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2873 Views: 5346
Edited by: Modesto Redrejo-Rodriguez
Reviewed by: Elizabeth LibbyChijioke Joshua
Original Research Article:
The authors used this protocol in Oct 2017
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Abstract
We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active hybrids were found to possess much fewer amino acid substitutions than the few hybrids that became inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Certain important residues that played important functional roles in mediating enzyme activity were identified. These findings suggest that DNA shuffling is an effective approach to identify and characterize important functional domains in bacterial proteins.
Keywords: CTX-M-14 CTX-M-15 DNA shuffling Hybrid enzyme Evolution
Background
DNA recombination is a natural process by which genetic materials are exchanged among bacteria to enhance survival fitness under environmental stresses. Several hybrid CTX-M-lactamases (CTX-M-64, CTX-M-123, CTX-M-137, and CTX-M-132), presumably resulting from recombination between the blaCTX-M-14 and blaCTX-M-15 genes, the most common variants worldwide, have been reported in recent years (Nagano et al., 2009; Tian et al., 2014; He et al., 2015; Liu et al., 2015). Among these hybrid enzymes, CTX-M-64, which contained the N- and C-terminal portions of CTX-M-15 and the middle fragment of CTX-M-14, exhibited even higher catalytic activity than their parental prototypes (He et al., 2015).
DNA shuffling is a molecular approach designed to mimic and accelerate the evolution process through PCR-mediated random combinations of two target genes (Crameri et al., 1998). Our previous study demonstrated the use of DNA shuffling to investigate the molecular events driving the evolution of the CTX-M-type β-lactamases (Po et al., 2017). Mutants with or without cefotaximase activity were recovered. Important amino acid residues that played a role in conferring enzyme activity were identified by comparative analysis of the genotypes and phenotypes of the mutants. Such approach can be employed to characterize other functional proteins. Here we describe a detailed protocol of in vitro DNA shuffling.
Materials and Reagents
96-well cell culture plate (SPL Life Sciences, catalog number: 30096 )
Test tubes
Cotton swab (HUBY-340-CA-006)
Filter (Pall, catalog number: 4612 )
Petri dish (Corning, GosselinTM, catalog number: SB93-101 )
E. coli DH5α (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12297016 )
E. coli BL21 (Thermo Fisher Scientific, InvitrogenTM, catalog number: C600003 )
pET15b
5x Green GoTaq® Flexi Reaction Buffer (Promega, catalog number: M8911 )
2.5 mM dNTP mixture (Takara Bio, catalog number: 4030 )
Magnesium chloride (UniChem, catalog number: M04550-4G )
Primers (Synthesized by BGI)
rTaq (Takara Bio, catalog number: R001WZ )
PBS (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10010023 )
1x TAE (see Recipes)
Milli-Q water
QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
DNase I (Sigma-Aldrich, catalog number: DN25-1G )
GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM1331 )
Gel loading dye (New England Biolabs, catalog number: B7024S )
SacI-HF (New England Biolabs, catalog number: R3156S )
BamHI-HF (New England Biolabs, catalog number: R3136S )
CutSmart® Buffer (New England Biolabs, catalog number: B7204S )
T4 DNA Ligase (New England Biolabs, catalog number: M0202S )
10x T4 DNA Ligase Buffer (New England Biolabs, catalog number: B0202S )
Tris (IBI Scientific, catalog number: IB70145 )
EDTA (Merck, catalog number: 1.08421.1000 )
Glacial acetic acid (DUKSAN, catalog number: 3839 )
Agarose, Molecular Biology Certified (IBI Scientific, catalog number: IB70045 )
10,000x Gold View I (West Gene, catalog number: WGO-1 )
LB Broth (Hopebio, catalog number: HB0128 )
LB Nutrient Agar (Hopebio, catalog number: HB0129 )
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-100G )
QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106 )
Isopropyl-β-D-thiogalactopyranoside, IPTG (Santa Cruz Biotechnology, catalog number: sc-202185B )
Mueller-Hinton broth, MH broth (BD, BBLTM, catalog number: 212322 )
Mueller-Hinton Agar, MH agar (Hopebio, catalog number: HB6232 )
Cefotaxime sodium salt (Sigma-Aldrich, catalog number: C7912-1G )
Sodium chloride (VWR, catalog number: VWRC27810.295 )
50 mM Magnesium chloride (see Recipes)
50x TAE buffer (see Recipes)
1% Agarose gel (see Recipes)
10x DNase I reaction mixture (see Recipes)
LB broth (see Recipes)
25 mg/ml Ampicillin (see Recipes)
0.5 M IPTG (see Recipes)
MH broth (see Recipes)
MH-IPTG (see Recipes)
MH agar (see Recipes)
Saline (see Recipes)
Equipment
Bio-Rad S1000TM thermal cycler (Bio-Rad Laboratories, model: S1000TM )
Centrifuge (Hettich Instruments, model: Mikro 185 )
Power pad for electrophoresis (Labnet International, model: ENDUROTM 300V , catalog number: E0303)
Gel tank
UV transilluminator
Spectrophotometer (Hach, model: DR 2800TM )
Conventional water bath (42 °C for transformation)
Conventional autoclave
Microwave
pH meter
Software
PyMOL software
BioEdit Sequence Alignment Editor
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Po, K. H. L., Chan, E. W. C. and Chen, S. (2018). Characterization of Protein Domain Function via in vitro DNA Shuffling. Bio-protocol 8(11): e2873. DOI: 10.21769/BioProtoc.2873.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Modification
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2,874 | https://bio-protocol.org/exchange/protocoldetail?id=2874&type=0 | # Bio-Protocol Content
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Peer-reviewed
3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
Jan Strnadel
SW Sang Myung Woo
SC Sunkyu Choi
HW Huawei Wang
MG Marian Grendar
KF Ken Fujimura
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2874 Views: 9432
Edited by: Chiara Ambrogio
Reviewed by: Mauro Sbroggio'Enrico Patrucco
Original Research Article:
The authors used this protocol in Apr 2017
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Apr 2017
Abstract
Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, spheroids cultured in hanging drops, magnetic levitation systems or 3D printing methods are already being widely used in research and for novel drug screening. In this article, we describe a detailed protocol for testing the effect of shRNA-mediated gene silencing on tumorsphere formation and growth. This approach allows researchers to test the impact of gene knockdown on the growth of tumor initiating cells. As verified by our lab, the protocol can be also used for isolation of 3D cancer cell lines directly from tumor tissues.
Keywords: 3D culture Tumorspheres Cancer cell line shRNA gene silencing
Background
3D in vitro cancer cell models represent a bridge experimental method between cell lines and tumors grown in vivo (Pampaloni et al., 2007; Weiswald et al., 2015). 3D characters of solid tumors with heterogeneous access to nutrients or oxygen can only be effectively mimicked by 3D culture systems. In recent years, protocols for tumorsphere culture gained lot of interest. A tumorsphere can be described as a solid, spherical object created from a single progenitor or stem cell. For tumorspheres formation assays, cells are seeded and grown in serum-free media in ultra-low attachment plates (non-adherent conditions), which allows enrichment of cancer cells with stem/progenitor properties (Johnson et al., 2013). Tumorspheres generated from freshly isolated tumor tissue are of special interest in the field because cells from established cell lines typically differ from the primary tumor due to mutations and abnormalities gained during multiple rounds of in vitro passaging. Hereby, we present an optimized protocol for 3D culture-based primary tumor cell isolation and the use of 3D culture to assess the effect of gene silencing on the growth of tumor-initiation cells.
Materials and Reagents
Eppendorf tube
Pipette tips
Vials
Plastic bottles
50 ml Falcon tube
Petri dishes with clear lid (Fisher Scientific, Fisherbrand, catalog number: FB0875712 )
6-well plates, Corning Costar Ultra-Low Attachment (Corning, catalog number: 3471 )
15 ml Falcon tubes (Corning, Falcon®, catalog number: 352196 )
70 µm cell filter (cell strainer - Corning, Falcon®, catalog number: 352350 )
Ice
Plastic pipette
24-well plates, Corning Costar Ultra-Low Attachment (Corning, catalog number: 3473 )
6-well plates (Corning, catalog number: 3506 )
Serological pipettes
Surgical razor blades (FisherBrand High Precision # 22 Style Scalpel Blade, Fisher Scientific, FisherbrandTM, catalog number: 12-000-161 )
Sterile pipets (10 ml) (FisherBrand Sterile Disposable Standard Serological Pipets, Fisher Scientific, FisherbrandTM, catalog number: 13-678-14A )
Cryovials (General Long-Term Storage Cryogenic Tubes 1 ml, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5000-1012 )
(optional) Mission pLKO.1-puro-CMV-TurboGFP Positive control Transduction Particles (Sigma-Aldrich, catalog number: SHC003V )
EDTA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575-020 )
PBS pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
Trypsin EDTA (0.05%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
SureEntry transduction reagent (QIAGEN, catalog number: 336921 )
Clorox Bleach (Veritiv, catalog number: 30966 )
Heparin Sodium Salt (Sigma-Aldrich, catalog number: H3149-10KU )
DMEM/F12, Glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 10565018 )
Penicillin/Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
B27 supplement 50x (serum free) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
bFGF – human Fibroblasts Growth Factor 147 basic (animal free) (Gemini Bio-Products, catalog number: 300-805P )
EGF– Epidermal Growth Factor (human) (Gemini Bio-Products, catalog number: 300-110P )
ROCK inhibitor (Y-27632) (STEMCELL Technologies, catalog number: 72304 )
Heparin solution (STEMCELL Technologies, catalog number: 07980 )
BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, catalog number: 356230 ) or Cultrex® 3-D Culture MatrixTM Reduced Growth Factor Basement Membrane Extract (Trevigen, catalog number: 3445-001-01 )
Notes:
Aliquot Matrigel or Cultrex Matrix into single-use aliquots in a sterile hood into sterile Eppendorf tubes. We recommend 0.5 ml of Matrigel per Eppendorf tube. When combined with 1.5 ml of ice-cold media, this amount is sufficient for 3D culture using one well in a six-well plate.
Use chilled pipettes and have Eppendorf tubes on ice during procedure.
Store at -80 °C. Thaw on ice or in the refrigerator overnight before use.
Hibernation medium (HibernateTM-A, Thermo Fisher Scientific, GibcoTM, catalog number: A1247501 )
Collagenase IV (Sigma-Aldrich, catalog number: C8051-100MG or similar)
Trypan Blue (TC-10, Bio-Rad Laboratories, catalog number: 1450013 )
Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240096 )
Dispase (1 U/ml, STEMCELL Technologies, catalog number: 07923 )
EDTA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
Accutase (STEMCELL Technologies, catalog number: 07920 )
DMSO (Sigma-Aldrich, catalog number: D2438-50ML )
Ethanol (70% solution, Fisher Scientific, Fisher BioReagentsTM, catalog number: BP8201500 )
Culture medium (see Recipes)
CSC medium (see Recipes)
Equipment
Centrifuge (temperature controlled, VWR® benchtop general purpose centrifuge) (VWR, catalog number: 10830-764 )
Pipettes
Forceps
CO2 Cell incubator (BINDER, model: CB 160 )
Biological Safety Level (BSL-2) laminar flow hood (Esco, EscoTechnologies, USA)
TC20 Automated cell counter (Bio-Rad)
Mr. Frosty Freezing Container (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5100-0001 )
FormaTM 7000 series Ultra Low Temperature Freezers (Thermo Fisher Scientific, Thermo ScientificTM, model: TFM#902 Series )
Liquid nitrogen storage tank
Nikon TE-2000E D-Eclipse Csi confocal microscope running Nikon Elements software (Nikon, model: Eclipse TE2000-E )
Software
Nikon microscope operating software
ImageJ or similar image processing software (https://imagej.nih.gov/ij/)
GraphPad Prism 6.0 software from GraphPad Software, USA
(https://www.graphpad.com/scientific-software/prism/)
R software (R Core Team, 2015 R: A language and environment for statistical computing)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Strnadel, J., Woo, S. M., Choi, S., Wang, H., Grendar, M. and Fujimura, K. (2018). 3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation. Bio-protocol 8(11): e2874. DOI: 10.21769/BioProtoc.2874.
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Category
Cancer Biology > Cancer stem cell > Cell biology assays
Cell Biology > Cell signaling > Intracellular Signaling
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2,875 | https://bio-protocol.org/exchange/protocoldetail?id=2875&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro Osteoclastogenesis Assays Using Primary Mouse Bone Marrow Cells
XZ Xueqian Zhuang
Guohong Hu
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2875 Views: 10712
Edited by: Ralph Thomas Boettcher
Reviewed by: Jalaj GuptaCody Kime
Original Research Article:
The authors used this protocol in Oct 2017
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Abstract
Osteoclasts are a group of bone-absorbing cells to degenerate bone matrix and play pivotal roles in bone growth and homeostasis. The unbalanced induction of osteoclast differentiation (osteoclastogenesis) in pathological conditions, such as osteoporosis, arthritis and skeleton metastasis of cancer, causes great pain, bone fracture, hypercalcemia or even death to patients. In vitro osteoclastogenesis analysis is useful to better understand osteoclast formation in physiological and pathological conditions. Here we summarized an easy-to-follow osteoclastogenesis protocol, which is suitable to evaluate the effect of different factors (cytokines, small molecular chemicals and conditioned medium from cell culture) on osteoclast differentiation using primary murine bone marrow cells.
Keywords: Osteoclastogenesis Osteoclast Primary bone marrow TRAP staining Cell culture
Background
The skeleton is maintained by successive and well-controlled absorbance and formation of bone mass during lifetime. In the bone cavity, each of these two activities is carried out by a specialized cell type: the bone-forming osteoblasts and bone-degrading osteoclasts. Osteoblasts and osteoclasts are derived from bone-resident mesenchymal cells and hematopoietic lineage progenitor cells, respectively. The differentiation of hematopoietic myeloid progenitor cells into mature osteoclasts are majorly controlled by receptor activator of nuclear factor-κB ligand (RANKL, encoded by TNFSF11) and macrophage colony-stimulating factor (M-CSF, encoded by CSF1) derived from osteoblasts and its progenitor cells (Suda et al., 1999). Unlike other cells, osteoclasts differentiate through fusion of a certain number of progenitor cells (Boyle et al., 2003). Thus, a key histological feature of mature osteoclasts is their multiple nuclei. After maturation, osteoclasts are capable of bone resorption by producing an acidified microenvironment to dissolve bone mass mainly composed of calcium phosphate, along with proteases to degrade extracellular matrix (Boyle et al., 2003). The dissolved bone matrix releases sequestered growth factors utilized by osteoblasts to expand their population (Kassem and Bianco, 2015). This cross-talk between osteoblasts and osteoclasts ensures coordinate bone-forming and -degenerating activity, which is dysregulated in a plethora of diseases, including osteoporosis, arthritis and bone metastasis of cancers (Rodan and Martin, 2000; Raisz, 2005; Gupta and Massague, 2006). Based on previous literature (Lu et al., 2009; Wang et al., 2014; Zhuang et al., 2017), here we describe a step-by-step protocol for an in vitro osteoclastogenesis assay using primary murine bone marrow cells that allows studying the effect of a broad range of factors/conditions (such as cytokines and conditioned medium) on osteoclast differentiation.
Materials and Reagents
Pipet tips (Autoclaved, any brand)
29 gauge syringe (BD, catalog number: 328421 )
40 µm nylon mesh cell strainer (Corning, catalog number: 352340 )
Minisart® NML syringe filter, 0.2 µm (Sartorius, catalog number: 17597-K )
14 mm round coverslips (any brand suitable for cell culture)
24 well plate and 100 mm Petri dish (Thermo Fisher Scientific, NuncTM, catalog numbers: 142475 and 172931 )
15 ml conical tubes (Corning, catalog number: 352196 )
Glass slides (OMANO, catalog number: OMSK-50PL )
Cell culture Petri dishes and multi-well plate (Thermo Fisher Science, catalog numbers: 174888 , 150350 , 142485 )
1.5 ml Eppendorf tubes (Fisher Scientific, catalog number: 05-408-129 )
Mouse aged between 4-7 weeks (any eligible provider, mouse strain/genetic background should be consistent with other assays)
α-MEM (Thermo Fisher Scientific, catalog number: A1049001 )
Fetal bovine serum (Thermo Fisher Scientific, catalog number: 10099141 )
Recombinant murine M-CSF (PeproTech, catalog number: 315-02 )
Recombinant murine RANKL (PeproTech, catalog number: 315-11 )
Red blood cell lysing buffer (BD, catalog number: 555899 )
Albumin, bovine serum, fraction V (Merck, catalog number: 12659 )
Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma-Aldrich, catalog number: 387A )
Acetone (Fisher Scientific, catalog number: A18-1 )
37% formaldehyde (Fisher Scientific, catalog number: BP531-500 )
Neutral Balsam (Sangon Biotech, catalog number: E675007 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S5136 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H9892 )
Culture medium (see Recipes)
Phosphate buffered saline (see Recipes)
Murine rRANKL and rM-CSF solution (see Recipes)
Equipment
Pipette (Eppendorf, sterilize surface by 70% ethanol)
Hemacytometer (OMANO, catalog number: OMSK-HEMA )
Dissecting tweezers and scissors (Autoclaved)
Scissors (Fisher Scientific, catalog number: 08-940 )
Tweezers (Fisher Scientific, catalog number: 12-460-612)
Manufacturer: Integra LifeSciences, Integra® Miltex®, catalog number: MH18782 .
Tweezers (Fisher Scientific, catalog number: 12-460-611)
Manufacturer: Integra LifeSciences, Integra® Miltex®, catalog number: MH18780 .
Cell culture incubator (any brand)
Centrifuge (Thermo Fisher Scientific, model: Hearus Labofuge 400 R )
Balance
Inverted microscope (Nikon Instruments, model: Eclipse Ti-S )
Water bath (any brand which can reach 37 °C)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhuang, X. and Hu, G. (2018). In vitro Osteoclastogenesis Assays Using Primary Mouse Bone Marrow Cells. Bio-protocol 8(11): e2875. DOI: 10.21769/BioProtoc.2875.
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Category
Developmental Biology > Cell growth and fate > Degeneration
Cell Biology > Cell isolation and culture > Cell differentiation
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2,876 | https://bio-protocol.org/exchange/protocoldetail?id=2876&type=0 | # Bio-Protocol Content
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Peer-reviewed
Preparation of Lipid-Stripped Serum for the Study of Lipid Metabolism in Cell Culture
AH Aaron M. Hosios
ZL Zhaoqi Li
EL Evan C. Lien
MH Matthew G. Vander Heiden
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2876 Views: 12951
Edited by: Ralph Thomas Boettcher
Reviewed by: Istvan StadlerTim Andrew Davies Smith
Original Research Article:
The authors used this protocol in Mar 2016
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Mar 2016
Abstract
Studying lipid metabolism in cultured cells is complicated by the fact that cells are typically cultured in the presence of animal serum, which contains a wide, variable, and undefined variety of lipid species. Lipid metabolism can impact cell physiology, signaling, and proliferation, and the ability to culture cells in the absence of exogenous lipids can reveal the importance of lipid biosynthesis pathways and facilitate the generation of media with defined lipid species. We have adapted a protocol to remove lipids from serum without eliminating its ability to support the proliferation of cells in culture. This method requires di-isopropyl ether and butanol and can be used to generate small batches of lipid-stripped serum in four days. The resulting serum supports proliferation of many cell lines in culture and can be used to compare the metabolism of cells in lipid replete and depleted conditions.
Keywords: Delipidation Lipid-depleted media Cell culture conditions Lipid-free serum Metabolism Proliferation
Background
Lipids are the major constituents of cell membranes that delineate biological compartments, and also play an important role in signaling pathways and energy storage (Baenke et al., 2013). Lipid metabolism is dysregulated in a variety of diseases, and recent studies have suggested that disrupting lipid biosynthesis may be an approach to inhibit tumor growth (Svensson and Shaw, 2016). Consequently, there is interest in studying the metabolic pathways that produce cellular lipid species. Culturing cells in media deprived of specific metabolic components can help provide a better understanding of how metabolic pathways support cell function. Standard culture media for most mammalian cells includes animal serum as a source of protein and growth factors (most commonly fetal bovine serum [FBS]). Animal serum contains a variety of lipid species within lipoprotein complexes and bound to serum albumin. The composition of animal serum is not identical across lots, and the nutrients levels in serum are not well defined in most experiments. Cells are able to obtain lipids from both de novo synthesis and from exogenous sources (Kamphorst et al., 2013; Hosios et al., 2016; Balaban et al., 2017), so the presence of serum lipids can complicate studies of de novo lipid metabolism. For example, although inhibitors of fatty acid synthase can inhibit proliferation, this effect is strengthened in lipid-depleted serum (Svensson et al., 2016). Microenvironments within the body likely vary in their lipid composition (Tourtellotte, 1959; Nanjee et al., 2000), suggesting that there are physiological cases where cells may be deprived of lipids.
In addition to serving as a source of lipids, serum also provides growth factors and other components that support the cell proliferation, and most cells cannot be studied for long periods of time in the absence of serum. Serum-free media formulations and lipoprotein-depleted sera are commercially available, but these are often expensive, are not optimized to support the growth of all cell lines, and lack a corresponding lipid-replete serum to serve as a control. To overcome these challenges, we have employed a bi-phasic extraction to remove lipids from FBS without denaturing serum proteins (Cham and Knowles, 1976). This method has been previously described on LipidomicNet, and we modified this approach to generate lipid-depleted serum and a corresponding lipid-replete serum from the same original FBS lot (Hosios et al., 2016). In our hands, some cells can be cultured in this serum for several passages without a substantial change in their proliferation rate, while other cells are more sensitive to the absence of lipids. We have also demonstrated increased de novo lipid synthesis in cells cultured with this lipid-depleted serum, indicating the expected metabolic response to this condition. This protocol provides an efficient way to remove lipids from a range of volumes of FBS, and generates both lipid-depleted serum and dialyzed, lipid-replete control serum for use in cell culture experiments.
Materials and Reagents
50 ml conical tubes (e.g., Corning, catalog number: 430829 )
0.2 µm low-protein binding filters (e.g., Thermo Fisher Scientific, catalog number: 566-0020 )
Slide-A-Lyzer dialysis cassettes (ThermoFisher), molecular weight cut-off ≤ 10 kDa
Plastic wrap (e.g., Saran wrap)
Glass serological pipettes
Fetal bovine serum (FBS)
Di-isopropyl ether (Sigma-Aldrich, catalog number: 296856 )
N-Butanol (Sigma-Aldrich, catalog number: 34867 )
Sodium chloride (Sigma-Aldrich, catalog number: 793566 )
Protein concentration assay (e.g., Bio-Rad Protein Assay, Bio-Rad Laboratories, catalog number: 5000006 )
Cholesterol assay (e.g., Sigma-Aldrich, catalog number: MAK043 )
Triglycerides assay (e.g., Thermo Fisher Scientific, catalog number: TR22421 )
Tissue culture media (e.g., DMEM and RPMI)
Saline solution at 4 °C (9 g/L sodium chloride, 154 mM) (see Recipes)
Equipment
Pipettes
Glass beakers
Glass graduated cylinders
Separating funnel (if preparing large volumes of serum)
Magnetic stir-plate
Chemical fume hood
Biological safety cabinet (level 2)
Tabletop centrifuge (capable of spinning 50 ml conical tubes at 4,000 x g)
Nitrogen gas source (industrial grade)
Spectrophotometer capable of measuring absorbance at the wavelength appropriate to the protein assay (e.g., 595 nm for Bio-Rad Protein Assay)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hosios, A. M., Li, Z., Lien, E. C. and Vander Heiden, M. G. (2018). Preparation of Lipid-Stripped Serum for the Study of Lipid Metabolism in Cell Culture. Bio-protocol 8(11): e2876. DOI: 10.21769/BioProtoc.2876.
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Category
Biochemistry > Lipid > Delipidation
Cell Biology > Cell metabolism > Lipid
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2,877 | https://bio-protocol.org/exchange/protocoldetail?id=2877&type=0 | # Bio-Protocol Content
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Peer-reviewed
Expansion of Airway Basal Cells and Generation of Polarized Epithelium
HL Hannah Levardon
LY Lael M. Yonker
BH Bryan P. Hurley
HM Hongmei Mou
Published: Vol 8, Iss 11, Jun 5, 2018
DOI: 10.21769/BioProtoc.2877 Views: 12383
Reviewed by: Meenal SinhaAntoine de Morree
Original Research Article:
The authors used this protocol in Oct 2017
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Abstract
Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies. Further, basal stem cells can be harvested from patients with a specific airway disease, such as cystic fibrosis, enabling investigation of potentially altered behavior of disease-specific cells in the appropriate context of the airway mucosa. Here we describe, in detail, a protocol for the serial expansion of airway basal stem cells to enable the generation of nearly unlimited airway basal cells that can be stored and readily available for subsequent culturing and differentiation. In addition, we describe culturing and differentiation of airway basal stem cells on permeable transwell filters at air-liquid interface to create functional mucociliary pseudostratified polarized airway epithelial mucosa.
Keywords: Airway basal stem cells Stem cell expansion Mucociliary differentiation on air-liquid interface (ALI) Pseudostratified airway epithelium BMP/TGFβ/SMAD signaling Airway diseases modeling
Background
Airway disease modeling and drug discovery have benefited greatly from the development and use of primary airway epithelial cultures grown on permeable transwell filters at air-liquid interface (ALI). This model has several advantages over immortalized cell lines in that the primary epithelial cells can differentiate into an airway mucosa that features multiple epithelial cell types including ciliated, serosal, and basal cells and their arrangement is quite reflective of in vivo cellular organization. Primary ALI models exhibit functional micro-physiological processes including beating cilia and the ability to secrete mucus, features which are notably absent in the cell line-derived epithelial monolayer. Further, primary cells do not rely on artificial immortalization or transformation, as cell line-derived epithelial cells do, and therefore are unencumbered by the potential deranged signaling seen in cell lines, which can misrepresent processes occurring in airway epithelium. Despite these notable limitations, immortalized cell lines are widely employed to model and investigate the airway epithelium because primary airway epithelial cells present their own set of challenges. Primary epithelial cells fail to replicate after a few passages and must be continuously harvested and isolated to complete each set of studies. In addition, molecular biology techniques to alter or delete the expression of genes of interest are difficult to achieve and sustain with primary epithelial cells. These disadvantages, creating both cost and technical hurdles, have hindered the widespread usage of primary ALI cultures despite their obvious advantages for investigating the airway mucosa.
Recently, transforming growth factor-β superfamily signaling (BMP/TGFβ/SMAD signaling) activity was found to be suppressed in p63+ basal cell compartments, but highly active in differentiated apically positioned cells (Mou et al., 2016). These observations facilitated the establishment of a novel culture platform with the use of dual SMAD inhibition to overcome the growth arrest and irreversible differentiation encountered in standard culturing of primary cells. Importantly, epithelial basal cell culture no longer relies upon co-culture of primary epithelial cells with mitotically inactive fibroblast feeder layers, a technique established in the 1970’s to enhance epithelial cell proliferation by improving the capacity of cultured cells to escape senescence (Rheinwald and Green, 1975). With this newly discovered ability to expand airway basal stem cells, patient-specific cells can be isolated and preserved from small biopsies of the airway, generating a virtually limitless supply of patient-specific airway epithelium in vitro (Mou et al., 2016). When differentiated on air-liquid interface, airway basal stem cell-derived epithelium forms a polarized mucociliary layer that exhibits proper epithelial architecture, physiology and response to clinically relevant pharmacologic agents (Mou et al., 2016; Yonker et al., 2017a and 2017b). Further, airway disease can be studied by generating basal stem cell-derived epithelium from individuals with airway disease, such as cystic fibrosis (CF), a genetic disease caused by a mutation in the CFTR gene, resulting in progressive respiratory failure. Primary ALI mucosa can be used to explore and understand cellular and molecular mechanisms that contribute to CF disease pathology.
Chronic infection and aberrant inflammation are hallmarks of CF (Yonker et al., 2015). Persistent infection with Pseudomonas aeruginosa, a typical pathogen in the CF airway, contributes to overzealous inflammation marked by pathological neutrophilic breach of the airway mucosa. A better understanding of cellular and molecular mechanisms mediating neutrophil trans-epithelial migration has the potential to inform and improve the efficacy of anti-inflammatory therapeutic strategies critically needed to treat inflammation mediated lung damage associated with CF. Recently, we have applied our advanced culture method in a co-culture system with neutrophils to investigate neutrophil-airway epithelium interaction and P. aeruginosa-induced neutrophil trans-epithelial migration (Yonker et al., 2017a and 2017b), Our findings from cultured human airway basal stem cells differentiated on ALI are consistent with prior molecular mechanistic studies implicating epithelial-derived neutrophil chemoattractant hepoxilin A3, an arachidonic acid metabolite synthesized by 12-lipoxygenase, as key in driving P. aeruginosa-induced trans-epithelial migration (Yonker et al., 2017a and 2017b). Additionally, these studies revealed previously unknown cellular mechanisms associated with neutrophilic breach of the airway mucosal barrier. Neutrophils applied to the basolateral aspect of the infected airway mucosal ALI organize in clusters before migrating through the mucosal barrier in response to epithelial infection. These organized clusters of neutrophils persist along the apical surface following trans-epithelial migration before individual neutrophils begin detaching from clusters into the airspace compartment (Yonker et al., 2017a). These results demonstrate that our expandable airway basal stem cell culturing and ALI differentiation system has tremendous potential to be exploited in a variety of ways to better understand micro-anatomy, physiology, interaction with pathogens, and innate immunity at the airway mucosa.
Below, we describe, in detail, a protocol for the generation of unlimited airway basal cells for the development of functional airway mucosal models that exhibit relevant physiological functioning. This protocol overcomes several barriers associated with investigations involving primary cells and represents a robust cellular platform for human disease modeling and drug development.
Materials and Reagents
For airway basal cell expansion
6-well tissue culture plate (Corning, Falcon®, catalog number: 353046 )
150 mm tissue culture-treated culture dish (Corning, catalog number: 430599 )
T175 culture flask (Thermo Fisher Scientific, catalog number: 159910 )
500 ml filter units (Merck, catalog number: SCGPU05RE )
804G cells
Note: 804G is a rat bladder epithelial cell line. It secretes matrix proteins enriched in laminin and collagen. The 804G-conditioned medium can be used as a cost-efficient coating medium.
Epithelial basal cell culture medium (Lonza, catalog number: CC-3118 or PromoCell, catalog number: C-21170 )
Airway culture medium LHC-9 (Thermo Fisher Scientific, GibcoTM, catalog number: 12680013 )
A8301 (antagonist of TGF/SMAD signaling, Tocris Bioscience, catalog number: 2939 )
DMH-1 (antagonist of BMP4/SMAD signaling, Tocris Bioscience, catalog number: 4126 )
CHIR99021 (agonist of WNT signaling, Tocris Bioscience, catalog number: 4423 )
Y27632 (ROCK Inhibitor, Tocris Bioscience, catalog number: 1254 )
Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
Penicillin-Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Dulbecco's phosphate-buffered saline (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
RPMI 1640 medium, GlutaMAXTM supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 61870036 )
Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16000-044 )
Complete Airway Basal Cell Culturing Medium (see Recipes)
For airway-liquid-interface differentiation
0.22 μm filter (EMD Millipore, catalog number: SLGP033RS )
6.5 mm2 Transwell® with 0.4 µm Pore Polyester Membrane Insert, Sterile (Corning, catalog number: 3470 ), packaged 12 inserts in a 24-well plate
12 mm2 Transwell® with 0.4 µm Pore Polyester Membrane Insert, Sterile (Corning, catalog number: 3460 ), packaged 12 inserts in a 12-well plate
PneumaCultTM-ALI Medium (STEMCELL Technologies, catalog number: 05001 )
Hydrocortisone (STEMCELL Technologies, catalog number: 07904 )
Heparin solution (STEMCELL Technologies, catalog number: 07980 )
Sodium chloride (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9759 )
100% ethanol (Sigma-Aldrich, catalog number: E7023 )
Complete ALI Medium (see Recipes)
Hydrocortisone Solution (see Recipes)
For immunofluorescence
Slides Thermofrost (Fisher Scientific, FisherbrandTM, catalog number: 12-550-15 )
FisherbrandTM Cover Glasses: Rectangles (Fisher Scientific, FisherbrandTM, catalog number: 12-544-E )
2 ml Eppendorf tubes (Eppendorf, catalog number: 022431048 )
ImmEdge Hydrophobic Barrier PAP Pen (Vector Laboratories, catalog number: H-4000 )
Fluoromount-G (SouthernBiotech, catalog number: 0100-01 )
DAPI (4',6-diamidino-2-phenylindole) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 62247 )
Triton X-100 (Sigma-Aldrich, catalog number: X100-500ML )
4% paraformaldehyde (Santa Cruz Biotechnology, catalog number: sc-281692 )
Bovine serum albumin (Sigma-Aldrich, catalog number: A2153-100G )
Sucrose (Sigma-Aldrich, catalog number: S7903 )
OCT compound (Fisher Scientific, catalog number: 23730571 )
Primary and secondary antibodies (see Table 1 below)
Table 1. Primary and Secondary antibodies for immunofluorescence
Catalog Number
Vendor
Name
Target
Primary antibody ab76013 Abcam Rabbit monoclonal, anti-NKX2.1, 1:200
Reacts with: Mouse, Rat, Human
Airway epithelium
Primary antibody
ab23630
Abcam
Rabbit polyclonal, anti-FOXA2, 1:200
Reacts with: Mouse, Rat, Human
Airway epithelium
Primary antibody
ab53121
Abcam
Rabbit polyclonal, anti-Cytokeratin 5, 1:500-1:1,000
Reacts with: Mouse, Rat, Human
Basal cells
Primary antibody
GTX102425
GeneTex
Rabbit polyclonal, anti-p63, 1:300
Reacts with: Mouse, Rat, Human
Basal cells
Primary antibody
T6793
Sigma-Aldrich
Mouse monoclonal (clone 6-11B-1), anti-Acetylated tubulin, 1:2,000-5,000
Reacts with: plant, pig, human, monkey, hamster, invertebrates, chicken, bovine, rat, frog, protista, mouse
Ciliated cells
Primary antibody
14-9965-82
Thermo Fisher Scientific
Mouse monoclonal (clone 2A5), anti-FOXJ1, 1:100
Reacts with: Mouse, Human
Ciliated cells
Primary antibody
HPA031828
Sigma-Aldrich
Rabbit polyclonal, anti-SCGB1A1 (CCSP), 1:200
Reacts with: Human
Club cells
Primary antibody
MA5-12178
Thermo Fisher Scientific
Mouse monoclonal (clone 45M1), anti-Mucin 5AC antibody, 1:200-500
Reacts with: Human
Goblet cells
Secondary antibody
A-21202 ; A-21203 ; A-31571
Thermo Fisher Scientific
Donkey anti-Mouse IgG (H+L), Alexa Fluor® 488, 594, 647 donkey anti-mouse IgG (H+L), 1:200
Mouse primary Ab
Secondary antibody
A-21206 ; A-21207 ; A-31573
Thermo Fisher Scientific
Donkey anti-Mouse IgG (H+L), Alexa Fluor® 488, 594, 647 donkey anti-rabbit IgG (H+L), 1:200
Rabbit primary Ab
Secondary antibody
A-11055 ; A-11058 ; A-21447
Thermo Fisher Scientific
Donkey anti-Mouse IgG (H+L), Alexa Fluor® 488, 594, 647 donkey anti-goat IgG (H+L), 1:200
Goat primary Ab
Equipment
250 ml bottles (Corning, PYREX®, catalog number: 1395-250 )
Incubator at 37 °C with 5% CO2 (CO2 water jacketed incubator, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3111 )
Pipettes (PIPETMAN L Starter Kit, Gilson, catalog number: F167350 )
Centrifuge with swing bucket rotor for 15 ml and 50 ml centrifuge tubes (Beckman Coulter, model: CS-6 )
Vacuum pump
Sterile laminar flow hood (The Baker Company, Sterilgard II )
Hemostat or tweezers
Scalpel
37 °C water bath (VWR, catalog number: 89501-468 )
4 °C cold room
Inverted Light microscope (Nikon, diaphot TMD)
Olympus Fluoview FV10i confocal microscope
Olympus IX81 inverted fluorescence microscope (Olympus, model: IX81 )
Software
ImageJ software (downloaded from https://imagej.nih.gov/ij/)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Levardon, H., Yonker, L. M., Hurley, B. P. and Mou, H. (2018). Expansion of Airway Basal Cells and Generation of Polarized Epithelium. Bio-protocol 8(11): e2877. DOI: 10.21769/BioProtoc.2877.
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Category
Stem Cell > Adult stem cell > Epithelial stem cell
Cell Biology > Cell isolation and culture > Cell growth
Cell Biology > Cell isolation and culture > Cell differentiation
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2,878 | https://bio-protocol.org/exchange/protocoldetail?id=2878&type=1 | # Bio-Protocol Content
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Peer-reviewed
In vitro Chaperone Activity Assay Using α-Amylase as Target Protein
Jeeshma Nambidi Parambath
Gayathri Valsala
Karthik Menon
Shiburaj Sugathan
Published: Jun 20, 2018
DOI: 10.21769/BioProtoc.2878 Views: 6613
Edited by: Dennis Nürnberg
Reviewed by: Sabu AbdulhameedPooja Saxena
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Abstract
Small heat shock proteins (sHSP) are stress proteins which are ubiquitously found in almost all living organisms. They function as molecular chaperones, which assist in protein folding during translation and in the prevention of irreversible protein aggregation under denaturing conditions. This protocol describes the use of α-amylase as target protein in assessing the chaperone activity of wild and mutant recombinant small heat shock proteins of Mycobacterium leprae. Chaperone activity of these proteins, along with α-crystallin, a standard sHSP was demonstrated using a new method employing their protective effect against heat denaturation of α-amylase from porcine pancreas. The regained enzymatic activity of the α-amylase was demonstrated on starch agar plates stained with Iodine-Potassium Iodide (I2-KI) solution.
Keywords: Small heat shock proteins sHSP18 α-amylase Chaperone assay Heat inactivation
Background
Heat shock proteins (HSPs) are a conserved group of proteins which are induced when cells are exposed to external stress including heat and cold stress. Most of the members in this group are functionally related and are involved in the protein folding and unfolding mechanism. Small heat shock proteins (sHSPs) are a subset of HSPs with a molecular size ranging from 12 to 43 kDa and a conserved C-terminal region, called ‘α-crystalline domain’. The sHSPs show ATP independent molecular chaperone activity by binding to partially unfolded proteins and preventing their complete denaturation. There are several methods for demonstrating in vitro chaperone activity of sHSPs, using various substrate proteins like RuBisCO (Goloubinoff et al., 1989), rhodanese (Mendoza et al., 1992), insulin (Farahbakhsh et al., 1995), lysozyme (Rozema and Gellman, 1996), malate dehydrogenase (Lee et al., 1997), citrate synthase (Grallert et al., 1998), xylose reductase (Rawat and Rao, 1998), sorbitol dehydrogenase (Marini et al., 2000) and luciferase (Bepperling et al., 2012) etc. In these assays, the protective activity of sHSPs or other molecular chaperones is demonstrated based on their efficiency in refolding and prevention of aggregation during heat or chemical denaturation. These substrates have different quaternary structures, different rates of folding, and different tendencies to undergo irreversible side reactions during denaturation and HSP assisted renaturation. It has also been shown that protection against heat-inactivated restriction enzymes like NdeI and SmaI can be used to demonstrate the chaperone activity of α-crystallin in vitro (Hess and FitzGerald, 1998; Santhoshkumar and Sharma, 2001). HSP18 is one of the major immunodominant antigens of Mycobacterium leprae, and has been functionally characterized as an sHSP (Lini et al., 2008). In this protocol, we describe a simple method to assay chaperone activity of small heat shock proteins using heat inactivated α-amylase. The efficiency of sHSPs in preventing heat denaturation of α-amylase is demonstrated on starch agar plates after staining with an I2-KI solution. The iodine reagent binds to the starch polymer to form a dark blue color in the starch agar plates. Clear halos surrounding each well with sample are indicative of active amylase enzyme, which digests the starch in the agar plate (Atlas et al., 1995). The advantage of this method is its simplicity and the empirical demonstration of results on starch agar plates.
Materials and Reagents
Conical flasks 250 ml (BOROSIL, catalog number: 4980021 )
Dialysis tubing cellulose membrane (14 kDa cut-off, Sigma-Aldrich, catalog number: D9652 )
Microcentrifuge tubes 1.5 ml, 2 ml, 200 µl (Tarsons, catalog number: 500010 )
Parafilm (HiMedia, catalog number: LA017 )
Petri dishes 100 mm, plastic (Tarsons, catalog number: 461030 ), 150 mm, glass (BOROSIL, catalog number: 3160081 )
Pipette tips 1000, 200, 10 µl (Tarsons, catalog numbers: 521020 , 521010 , 520010 )
Polypropylene columns (6 ml) (QIAGEN, catalog number: 34924 )
Test tubes (BOROSIL, catalog number: 9820U04 )
Whatman No.1 filter paper (GE Healthcare, Whatman, catalog number: 1001090 )
96-well polystyrene flat-bottomed microplate (Tarsons, catalog number: 941196 )
Strains: E. coli strain C41 DE3 (pLysS: F – ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CmR) Lucigen was utilized for recombinant protein expression (Dumon-Seignovert et al., 2004)
Vector: pQE31 (QIAexpress Type IV Kit, QIAGEN, catalog number: 32149 )
Molecular chaperones: α-crystallin from bovine eye lens (Sigma-Aldrich, catalog number: C4163 ), recombinant wild and mutant sHSP18 of M. leprae (Lini et al., 2008)
Sterile water (Distilled)
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
Isopropyl-β-D-thiogalactoside (IPTG) (Sigma-Aldrich, catalog number: I6758 )
Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876 )
α-amylase enzyme from porcine pancreas (Sigma-Aldrich, catalog number: A3176 )
Ethanol (70%)
Starch soluble (HiMedia Laboratories, catalog number: GRM3029 )
Bradford reagent (Bio-Rad protein assay dye reagent concentrate, Bio-Rad Laboratories, catalog number: 5000006 )
Tryptone (HiMedia Laboratories, catalog number: RM9111 )
Yeast extract (HiMedia Laboratories, catalog number: RM027 )
Agar powder (HiMedia Laboratories, catalog number: GRM026P )
Sodium chloride (NaCl) (HiMedia Laboratories, catalog number: GRM3954 )
di-Sodium hydrogen phosphate dehydrate (Na2HPO4·2H2O) (HiMedia Laboratories, catalog number: RM257 )
Potassium dihydrogen orthophosphate, monobasic (KH2PO4) (HiMedia Laboratories, catalog number: GRM249 )
Sodium dihydrogen phosphate, monohydrate (NaH2PO4·H2O) (HiMedia Laboratories, catalog number: GRM3963 )
Potassium chloride (KCl) (HiMedia Laboratories, catalog number: RM698 )
Imidazole (HiMedia Laboratories, catalog number: RM1864 )
Potassium iodide (KI) (HiMedia Laboratories, catalog number: GRM1086 )
Iodine (HiMedia Laboratories, catalog number: GRM1064 )
TGX FastCast Acrylamide Kit (12%) (Bio-Rad Laboratories, catalog number: 1610175 )
Lysogeny broth (LB) (see Recipes)
Phosphate buffer saline (PBS, pH 7.4) (see Recipes)
Lysis buffer (see Recipes)
Starch agar (see Recipes)
Starch solution (see Recipes)
Iodine-potassium iodide (I2-KI) solution (see Recipes)
Equipment
Analytical balance (Sartorius, model: BSA423S )
Autoclave (Labline, India)
Cooling Centrifuge (HERMLE Labortechnik, model number: Z 323 K )
Deep freezer -80 °C (Labline, model: Ultrafreez )
Digital camera (Nikon, model: D5100 )
Incubator (Thermo Scientific Heratherm, model number: IGS60 )
Laminar air flow system (Genesys, model: GS LAF 4X2 )
Magic Mixer (TOPSCIEN, model: TMM-5L )
Magnetic stirrer (IKA, model: C-MAG HS 7 )
Cork-borer, nickel-plated brass, 6 mm diameter (Sigma-Aldrich, model number: Z165220 )
Milli-Q Integral Water Purification System for Ultrapure Water (Merck, catalog number: ZRXQ003WW )
Pipettes (1,000 µl, 100 µl, 10 µl and 2.5 µl) (Eppendorf, model: Research® plus )
SDS-PAGE Electrophoresis apparatus (Bio-Rad Laboratories, model: Mini-PROTEAN® 3 Cell )
Spectrophotometer (JASCO, model: V-730 )
Water bath (DAIHAN LABTECH, model number: LWB 211D )
Microplate reader (Tecan Trading, model: Infinite® M200 PRO )
Procedure
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Category
Biochemistry > Protein > Activity
Microbiology > Microbial biochemistry > Protein
Molecular Biology > Protein > Protein-protein interaction
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2,879 | https://bio-protocol.org/exchange/protocoldetail?id=2879&type=0 | # Bio-Protocol Content
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Peer-reviewed
Enhancement of Mucus Production in Eukaryotic Cells and Quantification of Adherent Mucus by ELISA
CR Christian Reuter
TO Tobias A. Oelschlaeger
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2879 Views: 9942
Edited by: David Cisneros
Reviewed by: Songjie CaiEmiel P.C. van der Vorst
Original Research Article:
The authors used this protocol in Nov 2017
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Nov 2017
Abstract
The mucosal surfaces of the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye harbor a resident microflora that lives in symbiosis with their host and forms a complex ecosystem. The protection of the vulnerable epithelium is primarily achieved by mucins that form a gel-like structure adherent to the apical cell surface. This mucus layer constitutes a physical and chemical barrier between the microbial flora and the underlying epithelium. Mucus is critical to the maintenance of a homeostatic relationship between the microbiota and its host. Subtle deviations from this dynamic interaction may result in major implications for health. The protocol in this article describes the procedures to grow low mucus-producing HT29 and high mucus-producing HT29-MTX-E12 cells, maintain cells and use them for mucus quantification by ELISA. Additionally, it is described how to assess the amount of secreted adherent mucus. This system can be used to study the protective effect of mucus, e.g., against bacterial toxins, to test the effect of different culture conditions on mucus production or to analyze diffusion of molecules through the mucus layer. Since the ELISA used in this protocol is available for different species and mucus proteins, also other cell types can be used.
Keywords: Mucus Mucin ELISA Cell culture HT29
Background
The interface of the body with the external environment is formed by mucosal surfaces. These mucosal epithelial tissues can be found for example in the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye. Due to their exposure to the external environment many microorganisms populate these tissues. Therefore, these epithelia have evolved multiple mechanisms of defense in response to their vulnerability to microbial attack. Many defensive compounds are secreted into the mucosal fluid, including mucins, antibodies, defensins, protegrins, collectins, cathelicidins, lysozyme, histatins, and nitric oxide (Kagnoff and Eckmann, 1997; Lu et al., 2002; Raj and Dentino, 2002).
To date, more than 20 genes encoding mucins have been identified in humans (Corfield, 2015). The human mucin (MUC) family comprises membrane-bound (MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15 – 17, MUC20 and MUC21) and secreted mucins (MUC2, MUC5AC, MUC5B, MUC6 – 9, MUC19) (Moran et al., 2011; Tailford et al., 2015).
The mucus layer of the intestinal epithelial surface is mainly composed of the secreted mucin MUC2, but the membrane-bound mucins MUC1, MUC3 and MUC4 are also expressed (Kim and Ho, 2010). In addition, the intestinal mucus layer differs in terms of composition, organization and thickness along the gastrointestinal tract (Tailford et al., 2015). The secreted mucins form a gel-like structure adherent to the apical cell surface that constitutes a physical and chemical barrier between the luminal contents and the underlying epithelium (Allan, 2011). Inflammasome activity controls the secretion of mucus in goblet cells and increased secretion of mucus is observed as the microbiota becomes more diverse (Jakobsson et al., 2015). It is becoming apparent that mucus plays a crucial role in maintaining a homeostatic balance between microbiota and its host. Even small deviations from this dynamic interaction can have significant health effects, among which are colitis, colorectal cancer and susceptibility to infection (McGuckin et al., 2011; Hansson, 2012; Chen and Stappenbeck, 2014).
In this protocol, we describe the culture of in vitro models producing different amounts of mucus depending on their culture condition (static vs. semi-wet with mechanical stimulation (Navabi et al., 2013). These models are based on the little to no mucus-producing HT29 cell line, a human colon adenocarcinoma cell line, and its high mucus-producing derivative HT29-MTX-E12 (E12). Furthermore, it is described how to quantify the mucus produced in the different models by ELISA. In addition, the mucolytic compound N-acetyl-L-cysteine (NAC) is used to remove adherent mucus in order to quantify the amount of secreted adherent mucus. A schematic overview of the workflow described in this protocol is provided in Figure 1.
The method described in this protocol is suitable to study the protective effect of mucus against bacterial toxins (Reuter et al., 2018), to test the effect of different culture conditions on mucus production (Navabi et al., 2013) or to analyze diffusion of molecules through the mucus layer. Since the ELISA used in this protocol is available for different species and mucus proteins, other cell types can also be used.
Materials and Reagents
1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352196 )
Absorbent paper
175 cm2 flask (Greiner Bio One International, CellStar®, catalog number: 660160 )
Transwell® Inserts with 0.4 µm porous polyester membranes and 12 mm diameter (Corning, Transwell®, catalog number: 3460 )
12-well plate (included in Corning Transwell® package; if additional plates are required: Corning, Costar®, catalog number: 3513 )
Cell lines HT29 (European Collection of Authenticated Cell Cultures (ECACC), catalog number: 91072201 ) and HT29-MTX-E12 (European Collection of Authenticated Cell Cultures (ECACC), catalog number: 12040401 ) or other cells to be tested for their mucus production
Deionized water
70% ethanol
Dulbecco’s modified Eagle’s medium (DMEM), high glucose, GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
Heat-inactivated fetal calf serum (FCS)
100x Penicillin/Streptomycin solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
100x Non-essential Amino Acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
Phosphate buffered saline (PBS), pH 7.0-7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
0.05% Trypsin-EDTA solution (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
ELISA kit for mucin (CLOUD-CLONE, catalog number: SEA705Hu )
Note: In this protocol, the ELISA kit SEA705Hu was used for measurement of human mucin 2 (MUC2). Kits for different species and mucus proteins are available at Cloud-Clone Corp., e.g., SEA413Mu for measurement of mouse mucin 1 (MUC1)).
N-acetyl-L-cysteine (Sigma-Aldrich, catalog number: A9165 )
Cell culture medium (see Recipes)
N-acetyl-L-cysteine working solution (see Recipes)
Equipment
Sterile forceps
Container for wash solution
Multichannel Pipette (volume range: 20-200 µl)
Water bath
Humidified CO2 incubator (Thermo Fisher Scientific, model: HeracellTM 150i )
Biological safety cabinet
Hemacytometer (BRAND, Neubauer Improved, catalog number: 717805 )
37 °C incubator (e.g., CO2 incubator at 37 °C with CO2 switched off)
Orbital shaker for use in CO2 incubators (Infors, model: Celltron )
Ultrasonicator (Ultrasonic Homogenizer, BioLogics, model: 300VT )
Microcentrifuge (Eppendorf, model: 5418 )
Swing Bucket Centrifuge (Thermo Fisher Scientific, model: HeraeusTM MegafugeTM 16R )
Microplate reader (Tecan Trading, model: Infinite® 200 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Reuter, C. and Oelschlaeger, T. A. (2018). Enhancement of Mucus Production in Eukaryotic Cells and Quantification of Adherent Mucus by ELISA. Bio-protocol 8(12): e2879. DOI: 10.21769/BioProtoc.2879.
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Category
Biochemistry > Protein > Immunodetection
Cell Biology > Cell-based analysis > Protein secretion
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288 | https://bio-protocol.org/exchange/protocoldetail?id=288&type=0 | # Bio-Protocol Content
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Peer-reviewed
Extraction of Coumarins from Leaves, Petioles, Stems and Roots of Ruta graveolens and Nicotiana benthamiana
AH Alain Hehn
GV Guilhem Vialart
AO Alexandre Olry
FB Frederic Bourgaud
Published: Vol 2, Iss 22, Nov 20, 2012
DOI: 10.21769/BioProtoc.288 Views: 11427
Original Research Article:
The authors used this protocol in May 2012
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May 2012
Abstract
This method describes the extraction of coumarins and furanocoumarins from leaves of Ruta graveolens (a natural furanocoumarin) producer, and Nicotiana benthamiana.
Materials and Reagents
Ruta graveolens or Nicotiana benthamiana plants
Liquid nitrogen
Ethanol
Methanol
(+)- Taxifolin (Extrasynthèse, catalog number: 1036 http://www.extrasynthese.com)
Equipment
2 ml microtubes
Centrifuge for 2 ml tubes
Pestle and mortar
Bench top blendar (Polytron PT2100, Kinematica, http://www.kinematica ch)
Vacuum concentrator (RC10.10 speed vacuum, http://www.thermoscientific.com)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hehn, A., Vialart, G., Olry, A. and Bourgaud, F. (2012). Extraction of Coumarins from Leaves, Petioles, Stems and Roots of Ruta graveolens and Nicotiana benthamiana. Bio-protocol 2(22): e288. DOI: 10.21769/BioProtoc.288.
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Category
Plant Science > Plant biochemistry > Other compound
Biochemistry > Other compound > Flavonoid
Plant Science > Plant physiology > Tissue analysis
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2,880 | https://bio-protocol.org/exchange/protocoldetail?id=2880&type=0 | # Bio-Protocol Content
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Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana
ML Mengqian Luo
XZ Xiaohong Zhuang
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2880 Views: 7461
Edited by: Zhibing Lai
Reviewed by: Yasin Dagdas
Original Research Article:
The authors used this protocol in Jan 2017
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Abstract
As a fundamental metabolic pathway to degrade and recycle cellular cargos, autophagy is highly induced upon stress, starvation and senescence conditions in plants. A double-membrane structure named autophagosome will form during this process for cargo sequestration and delivery into the vacuole.
A number of regulators have been characterized in plants, including the autophagy-related (ATG) proteins and other plant-specific proteins. Among them, ATG8 will undergo a lipidation process to become a membrane-bound ATG8-phosphatidylethanolamine form and mark the growing autophagosomal membrane as well as the completed autophagosome. Therefore, ATG8 has been regarded as a marker for autophagosomes; and biochemical detection of the membrane-associated form of ATG8 is used as one of the principal methods for measurement of autophagic activity. Here, we describe an ATG8 lipidation assay for detection of the ATG8-PE form using Arabidopsis thaliana seedlings.
Keywords: ATG8 ATG8-PE Lipidation Autophagy Autophagosome
Background
Autophagy is an essential metabolic process which mediates the bulk degradation of the damaged organelles and unwanted cellular contents. During autophagy, a double-membrane structure called autophagosome will form and deliver the cargos into the vacuole for degradation. The autophagy-related (ATG) proteins are required to regulate the autophagic activity (Liu and Bassham, 2012). Among them, two conjugation systems, including ATG8 conjugate and ATG5-ATG12 conjugate, are involved for autophagosome formation. Upon autophagic induction, the ATG5-ATG12 conjugate forms and functions as an E3-like enzyme to promote ATG8 lipidation for binding to the phosphatidylethanolamine (PE) on the autophagosome membrane (Ohsumi, 2001). Although ATG8-PE on the outer membrane will be recycled before the autophagosome fusion with the vacuole, ATG8-PE on inner membrane will traffic together with the cargo into the vacuole for degradation. Thus, the amount of ATG8-PE usually correlates with the number of punctate ATG8-positive structures as well as autophagic activity (Mizushima et al., 2010). Particularly, due to the high hydrophobicity of ATG8-PE, ATG8-PE migrates faster than ATG8 in SDS-PAGE gel, though the actual molecular weight of ATG8-PE is larger than the unconjugated ATG8 (Mizushima and Yoshimori, 2007). Accordingly, the amount of ATG8-PE from cell membrane fraction (CM) can be detected by immunoblotting with ATG8 antibodies. For example, in Arabidopsis atg5 mutant, the level of ATG8-PE is severely impaired upon autophagic induction, whereas no autophagosome structures labeled by ATG8 are formed (Chung et al., 2010). Therefore, biochemical detection of the ATG8 lipidation can serve as a useful method to access the autophagic activity when combined with different treatments, which has been applied in our previous study as well as others related to plant autophagy (Chung et al., 2010; Suttangkakul et al., 2011; Li et al., 2014; Zhuang et al., 2017). Here, we describe the protocol for ATG8 lipidation detection by ultracentrifuge separation of the membrane and cytosol fractions using acibenzolar-S-methyl (BTH)-treated seedlings (Zhuang et al., 2017).
Materials and Reagents
10 µl pipette tips (Thermo Fisher Scientific, catalog number: 3510 )
200 µl pipette tips (Wolf Laboratories, catalog number: 2100.YN )
1,000 µl pipette tips (Thermo Fisher Scientific, catalog number: 3580 )
1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
PVDF membrane
X-ray film (Advansta, catalog number: L-07014-100 )
5-day-old seedlings
MS salt (Caisson, catalog number: MSP01-50LT )
UltraPureTM Sucrose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15503022 )
BTH (Acibenzolar-S-methyl) (Sigma-Aldrich, catalog number: 32820 )
Methanol (VWR, BDH, catalog number: 10158 )
Liquid nitrogen
PIC (Protease Inhibitor Mixture) (Roche Diagnostics, catalog number: 11873580001 )
Tris Base (Caisson, catalog number: T041-1KG )
NaCl (Alfa Aesar, USB, catalog number: J21618 )
EDTA (Alfa Aesar, USB, catalog number: J15701 )
SDS (Sodium dodecyl sulfate) (Alfa Aesar, USB, catalog number: J75819 )
Triton X-100 (GE Healthcare, Amerrsham, catalog number: 17-1315-01 )
Glycerol ultrapure (Alfa Aesar, USB, catalog number: J16374 )
Bromophenol Blue (Sigma-Aldrich, catalog number: B5525 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Urea (Alfa Aesar, USB, catalog number: J75826 )
40% Acrylamide/Bis Solution (Bio-Rad Laboratories, catalog number: 161-0148 )
TEMED (Bio-Rad Laboratories, catalog number: 161-0801 )
APS (Ammonium persulfate) (USB, catalog number: US76322 )
Non-fat milk powder
ATG8 antibody (Agrisera, catalog number: AS14 2769 )
cFBPase (Agrisera, catalog number: AS04 043 )
Secondary antibody (Anti-rabbit IgG peroxidase conjugate, Sigma-Aldrich, catalog number: A6154 )
Precision Plus ProteinTM Dual Color Standards (Bio-Rad Laboratories, catalog number: 161-0374 )
Sodium hydrogen carbonate (NaHCO3) (VWR, catalog number: 144-55-8 )
Sodium carbonate (Na2CO3) (USB, catalog number: 21602 )
Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (USB, catalog number: 20233 )
Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (Sigma-Aldrich, catalog number: 04272 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 31248 )
Tween-20 (Sigma-Aldrich, catalog number: 63158 )
MS liquid medium (see Recipes)
10 mM BTH stock (see Recipes)
25x PIC (see Recipes)
10% (v/v) Triton X-100 (see Recipes)
1 M Tris-HCl stock (pH 6.8 or pH 7.4) (see Recipes)
0.5 M EDTA (pH 8.0) (see Recipes)
5x extraction buffer (see Recipes)
1x extraction buffer containing 1x PIC and 1% (v/v) Triton X-100 (see Recipes)
5x sample loading dye (see Recipes)
30% APS (see Recipes)
3x separation buffer (see Recipes)
5x stacking buffer (see Recipes)
15% SDS-PAGE gel with 6 M urea (see Recipes)
15% Urea separating gel
5% Stacking gel
Running buffer (see Recipes)
Transfer buffer (see Recipes)
PBS (see Recipes)
PBS-T (see Recipes)
Equipment
Eppendorf Research® plus Pipette 0.5-10 µl (Eppendorf, catalog number: 3120000020 )
Eppendorf Research® plus Pipette 10-100 µl (Eppendorf, catalog number: 3120000046 )
Eppendorf Research® plus Pipette 100-1,000 µl (Eppendorf, catalog number: 3120000062 )
Mortar and pestle
Centrifuge (Eppendorf, model: 5430 )
X-ray film cassette (Amersham Biosciences HypercassetteTM)
Ultracentrifuge tube (7 x 20 mm)
Ultracentrifuge (Beckman Coulter, model: OptimaTM MAX-XP )
Western Blotting apparatus (Bio-Rad)
Developer machine (Fujifilm FPM100A)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Luo, M. and Zhuang, X. (2018). Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana. Bio-protocol 8(12): e2880. DOI: 10.21769/BioProtoc.2880.
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Category
Plant Science > Plant immunity > Host-microbe interactions
Molecular Biology > Protein > Detection
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2,881 | https://bio-protocol.org/exchange/protocoldetail?id=2881&type=0 | # Bio-Protocol Content
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Peer-reviewed
Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies
Mayuri Sadoine
MC Michele Cerminara
Jörg Fitter
Alexandros Katranidis
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2881 Views: 5722
Edited by: Elizabeth Libby
Reviewed by: Kate Hannan
Original Research Article:
The authors used this protocol in Jan 2018
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Abstract
Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET studies. The protocol describes two cell-free approaches to achieve a selective label scheme that allows the highest possible accuracy in inter‐dye distance determination.
Keywords: Cell-free protein synthesis Selective double labeling Unnatural amino acids Precharged tRNA Single-molecule FRET
Background
Single-molecule FRET (smFRET) is one of the most prominent tools in structural biology, in particular for analyzing structures and functional conformational changes of proteins (Michalet et al., 2006; Roy et al., 2008; Sustarsic and Kapanidis, 2015). However, an extensive application of smFRET is in many cases limited by the elaborative production of suitable protein samples. These proteins need to be equipped with two fluorophores, site-specifically attached at different positions within the protein structure.
The classical cell-based production of proteins requires a sequence of time-consuming steps that can be overcome by employing cell-free protein synthesis (CFPS) systems, allowing a much faster and straightforward production and selection of proper double-labeled proteins. Moreover, another advantage of CFPS is given by the fact that several classes of proteins like proteases or membrane proteins, which are toxic to living cells or for other reasons are difficult to express in cells, can be synthesized successfully in CFPS systems. Finally, CFPS is an ideal tool for labs focusing on spectroscopic techniques like smFRET, since cell cultures are not necessary and safety regulations for recombinant organisms do not have to be considered.
Despite the inherently low sample amounts required for smFRET, CFPS was so far not standardly employed for the production of samples in smFRET studies. This was mostly due to the much lower protein yields as compared to cell-based systems and the lack of an appropriate cell-free approach that allows for a convenient synthesis of adequate amounts of double-labeled protein. However, as demonstrated by our group, thanks to either a much more efficient orthogonal label scheme (Sadoine et al., 2017) or a direct incorporation of dyes with superior photophysical properties (Sadoine et al., 2018), the obtained protein yield from our two cell-free approaches perfectly meets the requirement of related measurements. Combined with state-of-the-art procedures in smFRET studies, the obtained efficiency histograms allow for the highest possible accuracy in inter-dye distance determination the method can deliver today.
Here we present a general combined protocol to be used as a guide for preparing double-labeled proteins suitable for smFRET studies, using either one of the two cell-free methods or a combination of both. CFPS and smFRET represent a perfect combination to achieve the full potential of analyzing protein structures with fluorescence-based techniques and the two presented approaches have the potential to become the standard method to produce protein samples for smFRET studies.
Materials and Reagents
Eppendorf epT.I.P.S. standard pipette tips (Eppendorf, catalog numbers: 022492004 , 022492047 , 022492055 )
Eppendorf Safe-Lock tubes 1.5 ml (Eppendorf, catalog number: 022363204 )
Eppendorf Protein LoBind tubes 1.5 ml (Eppendorf, catalog number: 022431081 )
Zeba Spin Desalting Columns, 7K MWCO, 0.5 ml (Thermo Fisher Scientific, catalog number: 89882 )
Coplin staining jar (DWK Life Sciences, Wheaton, catalog number: 900470 )
High precision coverslides thickness No. 1.5H (Paul Marienfeld GmbH, catalog number: 0107242 )
RTS 100 Site-specific Label Kit (biotechrabbit, catalog number: BR1402601 )
Customized Cell-free E. coli-based protein synthesis system (RF1-depleted, w/o Cys) (biotechrabbit)
SUPERase•InTM RNase inhibitor (Thermo Fisher Scientific, catalog number: AM2694 )
cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 11836170001 )
Ni-NTA Magnetic Agarose Beads, 5% suspension (QIAGEN, catalog number: 36111 )
Alexa Fluor 488 C5-maleimide (Thermo Fisher Scientific, catalog number: A10254 )
Click-IT Alexa Fluor 647 DIBO alkyne (Thermo Fisher Scientific, catalog number: C10408 )
Atto633-AF-tRNACUA, amber (ProteinExpress, catalog number: CLD03 )
BODIPY FL-Cys-tRNACys, cysteine (biotechrabbit, custom made)
3-aminopropyltriethoxysilane (APTES) (Sigma-Aldrich, catalog number: 440140 )
Uvasol acetone (Merck, catalog number: 100022 )
Uvasol methanol (Merck, catalog number: 106002 )
Optical glue Norland Optical Adhesive 81 (Norland Products, catalog number: NOA 81 )
Nitrogen 5.0 gas bottle 99.999% purity, 50 Lt, 200 bar (Linde Gas)
Sodium bicarbonate (NaHCO3) for molecular biology (Sigma-Aldrich, catalog number: S5761 )
Methoxy-PolyEthyleneGlycol-Succinimidyl-active ester (PEG-NHS) (Rapp Polymere, catalog number: 125000-35 )
Phosphate-Buffered Saline (PBS) 10x pH 7.4 for molecular biology (Thermo Fisher Scientific, catalog number: AM9625 )
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (Thermo Fisher Scientific, PierceTM, catalog number: 20490 )
3-(N-Morpholino)propanesulfonic acid (MOPS) buffer grade (AppliChem, catalog number: A1076,1000 )
Sodium chloride (NaCl) for molecular biology (Sigma-Aldrich, catalog number: S3014 )
Imidazole buffer grade (AppliChem, catalog number: A1073,1000 )
Vaprox Hydrogen peroxide (H2O2) 35% (STERIS, catalog number: PB006 )
Sulfuric acid (H2SO4) ACS reagent (Sigma-Aldrich, catalog number: 258105 )
Lysis buffer (see Recipes)
Wash buffer (see Recipes)
Elution buffer (see Recipes)
Final buffer (see Recipes)
Labeling buffer azide (see Recipes)
Labeling buffer maleimide (see Recipes)
Piranha solution (see Recipes)
Equipment
Reverse action tweezers (IDEAL-TEK, catalog number: 2AX.SA )
Duran 250 ml beakers (DWK Life Sciences, Duran, catalog number: 21 106 36 )
Tubing cutter (BOLA, catalog number: S1852-28 )
pH-meter S20 SevenEasy with InLab Micro Electrode (Mettler-Toledo International, model: S20 , product discontinued, newer models can be used)
Eppendorf Thermomixer Compact (Eppendorf, catalog number: 5384000020 )
Eppendorf Refrigerated Microcentrifuge (Eppendorf, model: 5417R )
Stuart Rotator SB2 (Carl Roth, catalog number: Y549.1 )
Vortex Genie 2 (Scientific Industries, model: Vortex-Genie 2, catalog number: SI-0256 )
NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 2000c , catalog number: ND-2000C)
Confocal microscope MicroTime 200 with an inverted Olympus IX-81 microscope, pulsed excitation, pinhole diameter 30-75 μm and simultaneous dual color detection (PicoQuant, model: MicroTime 200 ; Olympus, model: IX81 )
HydraHarp 400 Time Correlated Single Photon Counting (TCSPC) Acquisition unit (PicoQuant, model: HydraHarp 400 )
Spectrofluorometer QuantaMaster 7 (Photon Technology International now Horiba, product discontinued, newer models can be used)
High Output Vacuum-Pressure Pump (Merck, catalog number: WP6222050 )
UV light transilluminator (Biometra, product discontinued, newer models can be used)
Software
MATLAB R2012a and later versions (The MathWorks Inc.)
SymPhoTime 64 (PicoQuant GmbH)
OriginPro 2018 (OriginLab Corporation)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sadoine, M., Cerminara, M., Fitter, J. and Katranidis, A. (2018). Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies. Bio-protocol 8(12): e2881. DOI: 10.21769/BioProtoc.2881.
Download Citation in RIS Format
Category
Biochemistry > Protein > Single-molecule Activity
Biochemistry > Protein > Fluorescence
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2,882 | https://bio-protocol.org/exchange/protocoldetail?id=2882&type=0 | # Bio-Protocol Content
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Peer-reviewed
Small Molecule-Based Retinal Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells
JZ Jie Zhu
DL Deepak A. Lamba
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2882 Views: 6847
Edited by: Giusy Tornillo
Reviewed by: Dongsheng Jiang
Original Research Article:
The authors used this protocol in Mar 2017
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The authors used this protocol in:
Mar 2017
Abstract
Retinal degeneration leads to loss of light-sensing photoreceptors eventually resulting in vision impairment and impose a heavy burden on both patients and the society. Currently available treatment options are very limited and mainly palliative. Ever since the discovery of human pluripotent stem cell technologies, cell replacement therapy has become a promising therapeutic strategy for these patients and may help restore visual function. Reproducibly generating enriched retinal cells including retinal progenitors and differentiated retinal neurons such as photoreceptors using human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in a dish is an essential first step for developing stem cell-based therapies. In addition, this will provide a reliable and sufficient supply of human retinal cells for studying the mechanisms of diseases. Here we describe a small molecule-based retinal induction protocol that has been used to generate retinal progenitors and differentiated retinal neurons including photoreceptors from several human ES and iPS cell lines. The retinal cells generated by this protocol can survive and functionally integrate into normal and diseased mouse retinas for several months following subretinal transplantation.
Keywords: Human ES Cells iPS Cells Retina Differentiation
Background
A number of groups around the world are developing methodologies to generate specific cell types from human pluripotent stem cells. These cells will likely play a critical role in the future of regenerative medicine as a source of replacement cells. These newly generated human cells will be very useful in developing better and more accurate human disease models that can then be used for discovery of novel drugs with better efficacy and safety profiles.
Our work focuses on retinal degenerative diseases such as macular degeneration and retinitis pigmentosa which affect millions of people worldwide. Death of light-sensing photoreceptors in the retina is commonly associated with those diseases and results in severe impairment or total loss of vision. There are no effective medical treatments available to cure those diseases.
Under specific conditions, human ES and iPS cells can be specifically used to generate retinal progenitor cells, and consequentially differentiate into specialized retinal neuronal subtypes (retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, and photoreceptors) (Osakada et al., 2008 and 2009; Hirami et al., 2009; Lamba et al., 2006, 2009 and 2010; Meyer et al., 2009; Hambright et al., 2012; Zhong et al., 2014). Establishment of effective and chemically-defined protocols to generate retinal progenitors as well as differentiated retinal cell types including photoreceptors from human ES cells and iPS cells is a critical step for developing cell replacement therapies for patients with a variety of incurable retinal degenerative diseases.
Here, we report a detailed small molecule-based retinal induction protocol that has been used to generate retinal cells in vitro and the derived retinal cells were used as donor cells in the transplantation studies carried out by Dr. Lamba’s research group. The derived retinal progenitors and retinal photoreceptors were tested in multiple host mouse lines with and without retinal degeneration conditions and showed the ability to survive and functionally integrate into the host mouse retina following transplantation (Zhu et al., 2017 and 2018).
Materials and Reagents
6-well plate (DNase and RNase free, treated) (Greiner Bio One International, catalog number: 657160 )
Cryogenic vials (Corning, catalog number: 430659 )
2 ml serological pipette (VWR, catalog number: 76093-882 )
1,000 µl micropipette (VWR, catalog number: 89079-974 )
15 ml conical tube (VWR, catalog number: 490001-623 )
Micropipette tip (VWR, catalog number: 490000-468 )
Cell scraper (Corning, catalog number: 3008 )
Sterile Disposable Filter System with PES Membrane (0.22 µm pore size) (Thermo Fisher Scientific, catalog number: 566-0020 )
Cover glasses (VWR, catalog number: 89015-725 )
70% ethanol (Sigma-Aldrich, catalog number: 459836 )
Matrigel (BD, BD Biosciences, catalog number: 354234 )
DMEM/F-12 basal medium (GE Healthcare, catalog number: SH30023.02 )
Essential 8 medium (Thermo Fisher Scientific, catalog number: A2858301 )
Penicillin Streptomycin Amphotericin B (Lonza, catalog number: 17-745E )
Lyophilized Y27632 (Stemgent, catalog number: 04-0012 )
1x Dulbecco’s phosphate buffered saline (DPBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190144 )
0.1% EDTA (Diluted 1x EDTA in DPBS at 1:1,000) (Corning, catalog number: 46-034-CI )
Sodium Pyruvate (Corning, catalog number: 25-000-Cl )
HEPES Buffer (Corning, catalog number: 25-060-CI )
Sodium Bicarbonate (Corning, catalog number: 25-035-CI )
Non-essential Amino Acid (Corning, catalog number: 25-025-CI )
N1 Supplement (Sigma-Aldrich, catalog number: N6530 )
Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
KnockOutTM Serum Replacer (KSR) (Thermo Fisher Scientific, catalog number: 10828028 )
IWR1-endo (Stemgent, catalog number: 04-0010 )
LDN 193189 (Stemgent, catalog number: 04-0074 )
Insulin-like growth factor 1 (IGF1) (R&D Systems, catalog number: 291-G1 )
TrypLE Express dissociation reagent (Thermo Fisher Scientific, catalog number: 12604021 )
1x HBSS (Corning, catalog number: 21-022-CM )
Dimethyl Sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-1 )
Poly-D-lysine (Sigma-Aldrich, catalog number: P6407 )
4% Paraformaldehyde solution (Electron Microscopy Sciences, catalog number: 157-4 )
10% Normal Donkey Serum Blocking Solution (Jackson Immuno Research Laboratories, catalog number: 017-000-001 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Anti-fading Fluoromount G mounting medium (Electron Microscopy Sciences, catalog number: 17984-25 )
4',6-diamidino-2-phenylindole (DAPI, Enzo Life Scienes, catalog number: ENZ-52404 )
Monoclonal mouse anti PAX6 antibody (DHSB, catalog number: PAX6 )
Polyclonal goat anti LHX2 antibody (Santa Cruz Biotechnology, catalog number: sc-19344 or sc-517243 )
Biotinylated polyclonal goat anti human OTX2 antibody (R&D Systems, catalog number: BAF1979 )
Polyclonal Rabbit anti CRX antibody (GeneTex, catalog number: GTX124188 )
Anti-Recoverin Antibody (EMD Millipore, catalog number: AB5585 )
Freezing Medium (NSC + 0.5% FBS Medium + 10% DMSO)
Essential 8 human ESC/iPSC culture medium (see Recipes)
Rock inhibitor Y-27632 (10 mM, 1,000x, Stemgent, catalog number: 04-0012 ) (see Recipes)
Neuronal Stem Cell Culture Medium (NSC) (see Recipes)
NSC + 0.5% FBS Medium (see Recipes)
ISLI + KSR Retinal Induction Medium (see Recipes)
Equipment
Vertical laminar flow hood certified for Level II handling of biological materials
Water bath
Incubator with humidity and gas control to maintain 37 °C and 95% humidity in an atmosphere of 5% CO2 in air
Note: The incubator needs to be able to adjust the O2 level, as required for Step A4i.
Low-speed centrifuge with a swinging bucket rotor (e.g., Beckman Coulter, model: GS-6 ) with an adaptor for plate holders
Pipette-aid with appropriate serological pipettes
Hemocytometer
Micropipette with appropriate tips
Mr. Frosty freezing container at room temperature (Thermo Fisher Scientific, catalog number: 5100-0001 )
-150 °C freezer or liquid nitrogen (LN2) vapor tank
-80 °C freezer
-20 °C freezer
Refrigerator (2-8 °C)
Inverted microscope
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhu, J. and Lamba, D. A. (2018). Small Molecule-Based Retinal Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. Bio-protocol 8(12): e2882. DOI: 10.21769/BioProtoc.2882.
Download Citation in RIS Format
Category
Stem Cell > Pluripotent stem cell > Cell differentiation
Stem Cell > Embryonic stem cell > Cell differentiation
Cell Biology > Cell isolation and culture > Cell differentiation
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2,883 | https://bio-protocol.org/exchange/protocoldetail?id=2883&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Immunohistochemical Identification of Human Skeletal Muscle Macrophages
Kate Kosmac
BP Bailey D. Peck
RW R. Grace Walton
JM Jyothi Mula
PK Philip A. Kern
MB Marcas M. Bamman
RD Richard A. Dennis
CJ Cale A. Jacobs
CL Christian Lattermann
DJ Darren L. Johnson
Charlotte A. Peterson
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2883 Views: 15728
Edited by: Andrea Puhar
Reviewed by: Stefano CiciliotYann Simon Gallot
Original Research Article:
The authors used this protocol in Jan 2019
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The authors used this protocol in:
Jan 2019
Abstract
Macrophages have well-characterized roles in skeletal muscle repair and regeneration. Relatively little is known regarding the role of resident macrophages in skeletal muscle homeostasis, extracellular matrix remodeling, growth, metabolism and adaptation to various stimuli including exercise and training. Despite speculation into macrophage contributions during these processes, studies characterizing macrophages in non-injured muscle are limited and methods used to identify macrophages vary. A standardized method for the identification of human resident skeletal muscle macrophages will aide in the characterization of these immune cells and allow for the comparison of results across studies. Here, we present an immunohistochemistry (IHC) protocol, validated by flow cytometry, to distinctly identify resident human skeletal muscle macrophage populations. We show that CD11b and CD206 double IHC effectively identifies macrophages in human skeletal muscle. Furthermore, the majority of macrophages in non-injured human skeletal muscle show a ‘mixed’ M1/M2 phenotype, expressing CD11b, CD14, CD68, CD86 and CD206. A relatively small population of CD11b+/CD206- macrophages are present in resting skeletal muscle. Changes in the relative abundance of this population may reflect important changes in the skeletal muscle environment. CD11b and CD206 IHC in muscle also reveals distinct morphological features of macrophages that may be related to the functional status of these cells.
Keywords: Skeletal muscle Macrophages Immune cells Immunohistochemistry CD68 CD11b CD206 Flow cytometry
Background
Macrophages are pleotropic immune cells capable of adapting to changes in the local microenvironment. Over the last several years, research has shown that macrophage phenotype is dynamic, existing on a continuum (Mosser and Edwards, 2008; Italiani and Boraschi, 2014; Martinez and Gordon, 2014). However, to date macrophage populations continue to be described using the restrictive M1 and M2 classifications. It is commonly accepted that these designations are an oversimplification of macrophage phenotype and represent opposite extremes of a continuum (Mosser and Edwards, 2008; Gordon et al., 2014; Italiani and Boraschi, 2014; Martinez and Gordon, 2014; Murray et al., 2014). M1 macrophages are classically activated, have pro-inflammatory functions and are involved in host responses to pathogens and tissue injury. M2 macrophages are alternatively activated, exhibit anti-inflammatory functions and are involved in wound healing and tissue repair. In addition to the functional definition of M1 and M2, cell surface markers have been identified to distinguish between these populations. Surface markers associated with M1 macrophages include CD40, CD64 and the co-stimulatory molecules CD80/CD86 (Lolmede et al., 2009; Ambarus et al., 2012), whereas M2 macrophages have been shown to express high levels of CD163, CD206 and galactose receptors (Lolmede et al., 2009; Ambarus et al., 2012; Roszer, 2015).
From tissue to tissue, macrophage populations are heterogeneous adopting different functional roles depending on the local environment (Gordon et al., 2014; Italiani and Boraschi, 2014). This, coupled with the macrophage continuum, has led to inconsistencies with regard to identification and nomenclature across fields and across species (Murray et al., 2014). Skeletal muscle macrophages have primarily been studied in rodent models of injury, where the M1 versus M2 macrophage classification has proven useful (Smith et al., 2008; Chazaud et al., 2009; Tidball and Villalta, 2010; Kharraz et al., 2013; Novak and Koh, 2013; Saclier et al., 2013b; Rigamonti et al., 2014; Tidball et al., 2014; Wang et al., 2014; Sciorati et al., 2016; Varga et al., 2016; Mackey and Kjaer, 2017). The skeletal muscle response to injury is characterized by highly orchestrated temporal processes. Initially, M1 macrophages phagocytize damaged skeletal muscle fibers and debris, followed by M2 macrophage-facilitated repair and regeneration (Chazaud et al., 2009; Tidball and Villalta, 2010; Kharraz et al., 2013; Saclier et al., 2013a; Tidball et al., 2014; Sciorati et al., 2016). Recent work nicely details fiber repair in human skeletal muscle in vivo, showing the presence of macrophages, using a pan-macrophage intracellular marker (CD68+), in regenerating zones along injured fibers (Mackey and Kjaer, 2017). Direct interaction between macrophages and satellite cells (Dumont and Frenette, 2013; Ceafalan et al., 2017; Du et al., 2017; Wehling-Henricks et al., 2018), and defects in skeletal muscle regeneration in the absence of macrophage participation (Arnold et al., 2007; Melton et al., 2016), highlight the necessity of these cells for skeletal muscle repair. In vitro, M1 macrophages promote skeletal muscle cell proliferation and M2 macrophages promote differentiation, suggesting that macrophages may play a role in skeletal muscle growth adaptations, as well as repair (Arnold et al., 2007; Saclier et al., 2013b).
Skeletal muscle is a highly adaptable tissue, able to respond to a wide range of external stimuli, such as exercise, inactivity, hormones and nutritional signals. In contrast to the clearly defined, strongly polarizing responses elicited by acute skeletal muscle injury, the role of tissue resident macrophages during less polarizing processes, such as responses to the aforementioned stimuli, is relatively unknown. Under non-damaging exercise conditions, animal studies report an increase in macrophage populations following aerobic and resistance exercise, linked to both metabolic and growth adaptations (DiPasquale et al., 2007; Ikeda et al., 2013). However, the mechanisms by which macrophages in skeletal muscle influence training adaptations remain to be explored. It has also been reported that resident human skeletal muscle macrophage abundance is affected by aging, obesity and diabetes (Przybyla et al., 2006; Hong et al., 2009; Varma et al., 2009; Tam et al., 2012; Fink et al., 2014; Reidy et al., 2017); however, the inconsistent use of macrophage markers across studies has made the interpretation of these findings difficult. Further, the applicability of the distinctive M1/M2 markers of polarized macrophages to tissue resident macrophages is unclear, as surface markers may not be mutually exclusive on resident macrophages under non-polarizing conditions (Italiani and Boraschi, 2014). Thus, there is a need in the field for a standardized, validated method for identifying and quantifying macrophages in human skeletal muscle. Establishing a simple and reproducible protocol for studying muscle macrophages will aide in the characterization of their role in muscle adaptations to various stimuli, independent of injury.
Although results from studies of skeletal muscle macrophages in animal models are informative, these studies often use macrophage markers that are not directly translatable for use in humans. Even when human homologs do exist, the same surface markers in mouse and rat skeletal muscles often identify different populations in human skeletal muscles, complicating the extrapolation of findings from rodent models to human studies (Murray et al., 2014). For example, CD68 is used as a pan-macrophage marker in humans and an M1 marker in mice. There is a need in the field for a standardized, validated method for identifying and quantifying macrophages in human skeletal muscle. Taking into account the limited mass of frozen muscle tissue available for analyses from human skeletal muscle biopsies (normally in the range of 100 mg), an immunohistochemical method is the most feasible approach to identifying and quantifying human skeletal muscle macrophage populations.
A variety of markers have been used to characterize human macrophages by flow cytometry. The most detailed studies have been performed utilizing peripheral blood mononuclear cells (PBMCs), artificially polarized to an M1 or M2 phenotype (Martinez et al., 2006; Ambarus et al., 2012; Iqbal, 2015). These in vitro studies characterize the expression of various marker combinations on M1 and M2 macrophages and provide a good starting point for choosing markers to identify macrophage populations in frozen human skeletal muscle tissue. CD14 has been identified as a monocyte marker, expressed mainly by macrophages but also neutrophils and dendritic cells (Table 1). In blood, CD14 co-staining with CD16 is used to stratify monocytes into three subsets: classical (CD14++/CD16-), intermediate (CD14++/CD16+) and non-classical (CD14+/CD16++) (Sprangers et al., 2016; Boyette et al., 2017). It is thought that classical monocytes give rise to tissue macrophages under homeostatic conditions; however, during an inflammatory insult all monocyte populations differentiate into macrophages (Italiani and Boraschi, 2014; Sprangers et al., 2016). In tissue, CD16 is predominantly used to identify NK cells, but is also expressed on neutrophils, granulocytes, dendritic cells and some macrophage populations (Table 1). CD11b is a commonly used marker and is expressed on subsets of lymphocytes and monocytes, these include natural killer (NK) cells, granulocytes and macrophages (Table 1). CD68 is expressed by cells in the monocyte lineage, including macrophages, and is the most commonly used macrophage marker in human skeletal muscle tissue (Table 1) (Stupka et al., 2001; Beaton et al., 2002; Peterson et al., 2003; Crameri et al., 2004; Przybyla et al., 2006; Crameri et al., 2007; Mahoney et al., 2008; Mikkelsen et al., 2009; Varma et al., 2009; Paulsen et al., 2010a; Paulsen et al., 2010b; MacNeil et al., 2011; Tam et al., 2012; Chistiakov et al., 2017; Mackey and Kjaer, 2017; Reidy et al., 2017). CD68 is a member of the lysosomal/endosomal-associated membrane glycoprotein (LAMP) family of proteins, which are mainly associated with the endosomal/lysosomal compartment. Though largely intracellular, CD68 can traffic to the cell surface. Of note, other cell types have been reported to express CD68, including hematopoietic cells, fibroblasts and endothelial cells (Table 1) (Kunisch et al., 2004; Gottfried et al., 2008; Paulsen et al., 2013; Chistiakov et al., 2017). CD206, the mannose receptor, is a well-accepted macrophage marker in skeletal muscle and is widely used to identify M2 macrophage subsets (Lolmede et al., 2009; Ambarus et al., 2012; Italiani and Boraschi, 2014; Roszer, 2015), although CD206 expression by other cell types (including satellite cells) has been reported (Table 1) (Jansen and Pavlath, 2006). M2 macrophages also express CD163 (Table 1) (Lolmede et al., 2009; Ambarus et al., 2012; Roszer, 2015). CD80 and CD86 are co-stimulatory molecules expressed by antigen presenting cells upon activation and have been used to identify M1 macrophage populations (Table 1) (Mosser and Edwards, 2008; Lolmede et al., 2009; Ambarus et al., 2012). Using three grams of discarded human hamstring muscle from patients undergoing anterior cruciate ligament (ACL) reconstruction surgery, we isolated and labeled mononuclear cells with antibodies against some of the markers described above (CD11b, CD14, CD16, CD86 and CD206) and performed multichannel flow cytometry. Due to the intracellular expression of CD68, we were not able to include CD68 in flow cytometry analyses. Mononuclear cells from skeletal muscle did not express CD16, but co-expressed the other 4 markers tested (Figures 1A-1E). Thus, human skeletal muscle macrophages have a ‘mixed’ phenotype, co-expressing both M1 (CD86) and M2 (CD206) cell surface markers (Figure 1D).
Table 1. Overview of monocyte and macrophage markers
Figure 1. Flow cytometry from discarded human hamstring muscle showing co-expression of both M1 and M2 macrophage markers. Mononuclear cells isolated from human skeletal muscle express A. Both pan-monocyte markers CD11b and CD14; B. Both the M2 macrophage marker, CD206, and the pan marker, CD11b; C. Both the M1 marker, CD86, and the pan marker CD11b; D. Both the M2 marker, CD206, and the M1 marker, CD86; E. Overlay of CD206+/CD86+ populations from panel D onto CD14/CD11b flow plot shown in panel A. This overlay shows that CD206+/CD86+ macrophages (denoted in dark blue) also express the pan-monocyte markers CD11b and CD14. Red boxes indicate cells that are double positive for the markers shown.
Using these 4 cell surface antibodies against CD11b, CD14, CD86 and CD206 that label skeletal muscle resident macrophages, we sought to develop a simple and reproducible immunohistochemical method for identification of macrophages in fresh frozen human skeletal muscle sections. CD68 is a commonly used pan-macrophage marker in human skeletal muscle. However, CD68 expression is predominantly intracellular, requiring permeabilization steps to perform immunohistochemistry (IHC). These permeabilization steps lead to inconsistent results and compromise staining with additional antibodies for cell surface markers. The cell surface localization of CD11b results in better morphological definition of macrophages and more consistent staining across samples than intracellular CD68 staining. Moreover, CD11b can readily be combined with CD206 and other cell surface markers for IHC. For these reasons, we compared CD11b and CD68 staining, and found CD11b to be comparable to CD68 as a pan-macrophage marker in human skeletal muscle (Figures 2A-2E; CD11b and CD68 antibodies cannot be used to label sections simultaneously for technical reasons, see General Note 12). Furthermore, distinct morphological features of muscle macrophages that may be related to functional status was revealed through CD11b and CD206 double IHC (Figures 8A-8C) (Durafourt et al., 2012; McWhorter et al., 2013). We describe here a detailed method for combined IHC using CD11b and CD206 antibodies on frozen human skeletal muscle sections. We also describe in detail our approach to quantifying macrophage subsets in non-injured skeletal muscle. We find the majority of human muscle macrophages are CD11b+/CD206+, whereas a small subset are CD11b+/CD206- (Figures 4A-4C). We were unable to obtain IHC results with antibodies against M1 cell surface markers (CD80 or CD86). Of note, anti-CD163 works well on frozen human skeletal muscle and can be used in place of CD11b and in combination with CD206 with this protocol (see General Note 13). Moreover, combining Pax7 (a satellite cell marker) IHC with CD206 shows very little co-expression, supporting the conclusion that CD206 co-localizes with CD11b in human skeletal muscle and is a valid macrophage marker. This protocol allows reproducible quantification of the relative abundance of CD11b+/CD206+ and CD11b+/CD206- macrophage populations in human skeletal muscle, which can be extended to human skeletal muscle adaptations, aging and disease, enabling comparison of results across studies, across labs and across diverse human populations.
Materials and Reagents
Pipette tips:
101-1,000 µl (USA Scientific, TipOne, catalog number: 1122-1832 )
0.1-10 µl (USA Scientific, TipOne, catalog number: 1120-3812 )
Gloves (VWR, catalog number: 82026-424 )
Nalgene Dewar (for liquid nitrogen) (Sigma-Aldrich, catalog number: F9401 )
Tri-Pour Polypropylene beaker (for cooling isopentane in liquid nitrogen) (VWR, catalog number: 89011-786)
Manufacturer: MEDEGEN MEDICAL PRODUCTS, catalog number: PB5935-400 .
MX35 Ultra Low-Profile blades for cryotomy (Thermo Fisher Scientific, catalog number: 3053835 )
Superfrost Plus slides (Fisher Scientific, catalog number: 12-550-15 )
Shandon Single Cytoslides (Thermo Fisher Scientific, catalog number: 5991056 )
Shandon Single Cytofunnel (Thermo Fisher Scientific, catalog number: 5991040 )
ImmEdge PAP pen (Vector Laboratories, catalog number: H-4000 )
1.5 ml microcentrifuge tubes (USA Scientific, catalog number: 1615-5599 )
15 ml conical tube (VWR, catalog number: 89039-666 )
24 x 50 mm, No. 1 coverglass (VWR, catalog number: 48393-081 )
Kimwipes (KCWW, Kimberly-Clark, catalog number: 34120 )
Cork stoppers for making muscle mounts (Fisher Scientific, catalog number: 07-782J )
PTFE (Teflon) coated stainless steel spatula (Fisher Scientific, catalog number: 21-401-50A)
Manufacturer: Saint-Gobain Performance Plastics, catalog number: D1069292 .
#10 curved blade disposable scalpel (Sklar Surgical Instruments, catalog number: 06-3310 )
Dumont #7 curved forceps (Fine Science Tools, catalog number: 11270-20 )
Cryo Tongs (Thermo Fisher Scientific, catalog number: 4000388 )
Tragacanth gum, powder (Sigma-Aldrich, catalog number: G1128-500G )
Fisher O.C.T compound (Fisher Scientific, catalog number: 23-730-571 )
Isopentane (2-methylbutane) (Merck, catalog number: MX0760-1 )
Liquid nitrogen (Scott Gross, catalog number: SG #347 )
Dry ice (Scott Gross, no catalog number available)
Ice cold acetone, stored at -20 °C (Fisher Scientific, catalog number: A18-4 )
Streptavidin/Biotin blocking kit (Vector Laboratories, catalog number: SP-2002 )
2.5% normal horse serum (NHS) (Vector Laboratories, catalog number: S-2012 )
50% 1x PBS (see 21 and Recipes)/50% glycerol mounting medium (glycerol - VWR, catalog number: BDH1172-1LP ) or Vectashield (Vector Laboratories, catalog number: H-1000 )
Antibodies for IHC, dilutions made with 2.5% normal horse serum or PBS (Table 2)
ImmPRESS-AP Anti-Mouse IgG (alkaline phosphatase) polymer detection kit (Vector Laboratories, catalog number: MP-5402 )
ImmPACT Vector Red Alkaline Phosphatase (AP) Substrate (Vector Laboratories, catalog number: SK-5105 )
Antibodies for multichannel flow cytometry (Table 3)
Table 2. Detailed IHC Antibody Information
Primary Antibody
Company
Catalog Number
Reactivity
Host /Isotype
Dilution/Diluent
CD68
Dako
M0814
Human/Mouse/Rat/Monkey
Mouse/IgG1
(1:100)/2.5% NHS
CD11b
Cell Sciences
MON1019
Human
Mouse/IgG1
(1:100)/2.5% NHS
Purified IgG1, κ
BD
555746
N/A
Mouse/IgG1
(1:500)/2.5% NHS
C206
R&D Systems
AF2534
Human
Goat/IgG (polyclonal)
(1:200)/2.5% NHS
CD163
Hycult Biotech
HM2157
Human/Monkey
Mouse/IgG1
(1:50)/2.5% NHS
Pax7
Developmental Studies Hybridoma Bank
Pax7
Human/Mouse/Rat
Mouse/IgG1
(1:100)/2.5% NHS
Biotinylated goat anti-mouse IgG1
Jackson ImmunoResearch
115-065-205
Mouse IgG1
Goat/N/A
(1:1,000)/2.5% NHS
ImmPRESS –AP
Vector Laboratories
MP-5402
Mouse IgG
N/A
Neat/no dilution
Biotinylated rabbit anti-goat IgG
Vector Laboratories
BA-5000
Goat
Rabbit/N/A
(1:500)/2.5% NHS
Streptavidin HRP (SA-HRP)
Thermo Fisher Scientific
S911
Biotin
N/A
(1:500)/2.5% NHS
Streptavidin Alexa Fluor 594 (SA-594)
Thermo Fisher Scientific S32356
Biotin
N/A
(1:200)/2.5% NHS
Superboost TSA Alexa Fluor 488 (TSA 488)
Thermo Fisher Scientific
B40953
HRP
N/A
(1:500)/2.5% NHS
ImmPACT Vector Red kit
Vector Laboratories
SK-5105
Alkaline Phosphatase
N/A
According to manufacturer’s instructions
Table 3. Detailed antibody information for multichannel flow cytometry
Antigen
Fluorophore
Laser
Filter/Bandpass
Ex (nm)
Em (nm)
Concentration
(µg/µl)
µl/106 cells
(500 µl total
volume)
Company
Catalog Number
CD11b
Alex Fluor 488
Blue (488)
530/30
490
525
0.4*
25
BioLegend
301317
CD14
Pacific Blue
Violet (407)
450/50
410
455
0.5
20
BioLegend
325615
CD16
VioGreen Violet (407)
Violet (407)
525/50
405
520
NA
50
Miltenyi Biotec
130-113-959
CD86
Phycoerythrin (PE)
Blue (488)
575/26
496
578
0.1*
25
BioLegend
305405
CD206
PerCP/Cy5.5
Blue (488)
695/40
482
695
0.1*
25
BioLegend
321121
LIVE/DEAD
Fixable Blue
UV (325)
450/50
350
450
NA
0.5
Thermo Fisher Scientific
L34961
Mouse IgG1, κ
Alex Fluor 488
Blue (488)
530/30
490
525 0.2*
50
BioLegend
400132
Mouse IgG1, κ
Pacific Blue
Violet (407)
450/50
410
455
0.5
20
BioLegend
400131
REA Control (S)
VioGreen Violet (407)
Violet (407)
525/50
405
520
NA
50
Miltenyi Biotec
130-104-608
Mouse IgG2b, κ
Phycoerythrin (PE)
Blue (488)
575/26
496
578
0.2
12.5
BioLegend
400311
Mouse IgG1, κ
PerCP/Cy5.5
Blue (488)
695/40
482
695
0.2*
12.5
BioLegend
400149
*Concentration is not specific and varies by batch
LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation (Thermo Fisher Scientific, catalog number: L34961 ) (Table 3)
UltraComp eBeads Compensation Beads (Thermo Fisher Scientific, catalog number: 01-2222-41 )
Polyurethane ice bucket (Fisher Scientific, catalog number: 02-591-45 )
1x phosphate-buffered saline (PBS) (see Recipes)
Deionized (DI) water
Sodium Chloride (NaCl) (VWR, catalog number: 97061-266 )
Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O) (VWR, catalog number: 200007-704)
Manufacturer: Acros Organic, catalog number: 206515000 .
Potassium phosphate monobasic (KH2PO4) (Aldon, catalog number: PP0730-500GR )
Sodium hydroxide 10 N (NaOH) (VWR, catalog number: BDH7247-1 )
Hydrochloric acid, 6 N (HCl) (VWR, catalog number: 97064-758 )
30% Hydrogen peroxide, ACS, Stabilized (VWR, catalog number: BDH7690-1 ) (see Recipes)
DAPI for staining cell nuclei (4',6-diamidino-2-phenylindole) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 ) (see Recipes)
Rabbit polyclonal anti-Laminin (Sigma-Aldrich, catalog number: L9393 ). Use at a dilution of 1:100 in 2.5% NHS
Equipment
Epifluorescent microscope with automated stage (ZEISS, model: Axio Imager M1 )
Cryostat (Thermo Fisher Scientific, model: HM525 NX )
P1000 pipetman (Gilson, catalog number: F123602 )
P10 pipetman (Gilson, catalog number: F144802 )
P2 pipetman (Gilson, catalog number: F144801 )
Glass Coplin jars (VWR, catalog number: 470175-194)
Manufacturer: VARIETY GLASS, catalog number: 674 .
Humidifying slide chamber (10 slide staining tray with black lid) (Electron Microscopy Sciences, catalog number: 71396-B )
Variable speed 2D rocker, 14 x 12 work surface (USA Scientific, catalog number: 2527-2000 )
4 °C refrigerator (Fisher Scientific, model: Isotemp Value Lab, catalog number: 17LREEFSA )
-80 °C freezer (Thermo Fisher Scientific, model: Revco® Elite Plus )
Class II Type A/B3 Biological safety cabinet (NuAire)
LSR II flow cytometer equipped with a 355 nm, 405 nm and 488 nm lasers (BD Biosciences)
Shandon Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific, catalog number: A78300003 )
Software
Image capture software (Zeiss, Zen blue)
Image processing software with an event count tool (Zeiss, Zen blue or Zen lite)
Prism7 or equivalent graphing software (GraphPad)
FlowJo v10 (FlowJo)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kosmac, K., Peck, B. D., Walton, R. G., Mula, J., Kern, P. A., Bamman, M. M., Dennis, R. A., Jacobs, C. A., Lattermann, C., Johnson, D. L. and Peterson, C. A. (2018). Immunohistochemical Identification of Human Skeletal Muscle Macrophages. Bio-protocol 8(12): e2883. DOI: 10.21769/BioProtoc.2883.
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Category
Immunology > Immune cell staining > Immunodetection
Immunology > Immune cell imaging > Epifluorescence Microscopy
Cell Biology > Tissue analysis > Tissue staining
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2,884 | https://bio-protocol.org/exchange/protocoldetail?id=2884&type=0 | # Bio-Protocol Content
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Peer-reviewed
Extraction and 16S rRNA Sequence Analysis of Microbiomes Associated with Rice Roots
JE Joseph Edwards*
CS Christian Santos-Medellín*
VS Venkatesan Sundaresan
*Contributed equally to this work
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2884 Views: 16579
Edited by: Joëlle Schlapfer
Reviewed by: Yang BaiFrancesco Dal Grande
Original Research Article:
The authors used this protocol in Feb 2015
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The authors used this protocol in:
Feb 2015
Abstract
Plant roots associate with a wide diversity of bacteria and archaea across the root-soil spectrum. The rhizosphere microbiota, the communities of microbes in the soil adjacent to the root, can contain up to 10 billion bacterial cells per gram of soil (Raynaud and Nunan, 2014) and can play important roles for the fitness of the host plant. Subsets of the rhizospheric microbiota can colonize the root surface (rhizoplane) and the root interior (endosphere), forming an intimate relationship with the host plant. Compositional analysis of these communities is important to develop tools in order to manipulate root-associated microbiota for increased crop productivity. Due to the reduced cost and increasing throughput of next-generation sequencing, major advances in deciphering these communities have recently been achieved, mainly through the use of amplicon sequencing of the 16S rRNA gene. Here we first present a protocol for dissecting the microbiota from various root compartments, developed using rice as a model. We next present a method for amplifying fragments of the 16S rRNA gene using a dual index approach. Finally, we present a simple workflow for analyzing the resulting sequencing data to make ecological inferences.
Keywords: Root microbiome Amplicon sequencing Rhizosphere Rhizoplane Endosphere
Background
Various plant root niches host different microbial communities (microbiota) originating from the soil (Bulgarelli et al., 2012; Lundberg et al., 2012; Edwards et al., 2015; Zarraonaindia et al., 2015; Wagner et al., 2016). Distinct microbiota acquired by each root niche likely have varying metabolic potential and may therefore impact the health of the host plant in different ways (Finkel et al., 2017). Bacterial and archaeal community composition in root-associated microbiota can be inferred through the use of 16S rRNA gene sequencing (Caporaso et al., 2012). The relatively low cost of sequencing now allows for comparative studies across plant species using datasets gathered by different research groups; however, small aberrations in specimen collection, sequencing, and analysis protocols may lead to large differences in the inferred microbial communities (Duvallet et al., 2017). We present this protocol detailing how to collect and analyze root microbiota from rice in an attempt to promote reproducibility across the plant microbiome field.
Materials and Reagents
Falcon 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
1.5 ml microfuge tubes (E&K Scientific Products, catalog number: 280150 )
1.5 ml non-stick microfuge tubes (Thermo Fisher Scientific, AmbionTM, catalog number: AM12450 )
0.2 ml PCR tubes (GeneMate, catalog number: 3235-00-210IS )
Filtered pipette tips (10, 200, 1,000 µl) (VWR, catalog numbers: 89168-750, 89140-936, 89168-754)
Manufacturer: Biotix, catalog number: BT10XLS3 , BT200 , BT1250 .
Gloves (Medline Industries, catalog numbers: large, MDS192086 ; medium, MDS192085 ; small, MDS192084 )
Qubit 0.5 ml assay tubes (Thermo Fisher Scientific, catalog number: Q32856 )
Single edge razor blade (Personna, catalog number: 94-115-71 )
Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9939 )
DNeasy PowerSoil kit (QIAGEN, catalog number: 12888-100 )
Primer 515F (GTGCCAGCMGCCGCGGTAA)
Primer 806R (GGACTACHVGGGTWTCTAAT)
HotStar High Fidelity DNA polymerase kit (QIAGEN, catalog number: 202602 )
Agencourt Ampure XP beads (Beckman Coulter, catalog number: A63880 )
Qubit dsDNA HS assay kit (Thermo Fisher Scientific, catalog number: Q32851 )
Ethanol 200 proof (Sigma-Aldrich, catalog number: E7023-500ML )
Agarose (Biotech Sources, catalog number: G01PD-500 )
DNA gel loading dye
NucleoSpin gel and PCR clean-up kit (MACHEREY-NAGEL, catalog number: 740609.250 )
NaCl (Fisher Scientific, catalog number: S271-1 )
KCl (Fisher Scientific, catalog number: P217-500 )
Na2HPO4 (Fisher Scientific, catalog number: S374-500 )
KH2PO4 (Fisher Scientific, catalog number: P285-500 )
Autoclaved phosphate buffered saline (PBS) solution (~100 ml/plant) (see Recipes)
Equipment
96 well magnetic plate (Alpaqua Engineering, catalog number: A001219R )
1.5 ml tube magnetic rack (Thermo Fisher Scientific, catalog number: MR01 )
Pipettes (2.5, 10, 200, 1,000 µl) (Thermo Fisher Scientific, FinnpipetteTM, catalog numbers: 4641010N , 4641030N , 4641080N , 4641100N )
Ultrasonic cleaning bath, 40 kHz (Branson, model: Branson 1800, catalog number: CPX-952-116R )
Dissection tools (scissors and forceps) (scissors: Bioseal, catalog number: KI011/50 ; forceps: Integra LifeSciences, Miltex, catalog number: 6-184 )
-80 °C freezer
Microcentrifuge (Eppendorf, model: 5417C )
Mini-Beadbeater-96 high-throughput cell disrupter (Bio Spec Products, catalog number: 1001 )
Electrophoresis gel unit (Bio-Rad Laboratories, catalog number: 1704468 )
Qubit fluorometer (Thermo Fisher Scientific, catalog number: Q33226 )
PCR thermal cycler (Bio-Rad Laboratories, model: T100TM Thermal Cycler , catalog number: 1861096)
Software
Python2 version 2.7.12
R version 3.4.3
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Edwards, J., Santos-Medellín, C. and Sundaresan, V. (2018). Extraction and 16S rRNA Sequence Analysis of Microbiomes Associated with Rice Roots. Bio-protocol 8(12): e2884. DOI: 10.21769/BioProtoc.2884.
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Category
Plant Science > Plant immunity > Host-microbe interactions
Microbiology > Community analysis > Metagenomics
Systems Biology > Genomics > Sequencing
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2,885 | https://bio-protocol.org/exchange/protocoldetail?id=2885&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect
BN Ben Niu
RK Roberto Kolter
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2885 Views: 8594
Edited by: Joëlle Schlapfer
Reviewed by: Meng Wu
Original Research Article:
The authors used this protocol in Mar 2017
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Original research article
The authors used this protocol in:
Mar 2017
Abstract
Besides analyzing the composition and dynamics of microbial communities, plant microbiome research aims to understanding the mechanism of plant microbiota assembly and their biological functions. Here, we describe procedures to investigate the role of bacterial interspecies interactions in root microbiome assembly and the beneficial effects of the root microbiota on hosts by using a maize root-associated simplified seven-species (Stenotrophomonas maltophilia, Ochrobactrum pituitosum, Curtobacterium pusillum, Enterobacter cloacae, Chryseobacterium indologenes, Herbaspirillum frisingense and Pseudomonas putida) synthetic bacterial community described in our previous work. Surface-sterilized maize seeds were grown in a gnotobiotic system based on double-tube growth chambers after being soaked in suspensions containing multiple species of bacteria. The dynamics of the composition of the bacterial communities colonized on maize roots were tracked by a culture-dependent method with a selective medium for each of the seven strains. The impact of bacterial interactions on the community assembly was evaluated by monitoring the changes of community structure. The plant-protection effects of the simplified seven-species community were assessed by quantifying (1) the growth of a fungal phytopathogen, Fusarium verticillioides on the surfaces of the seeds and (2) the severity of seedling blight disease the fungus causes, in the presence and absence of the bacterial community. Our protocol will serve as useful guidance for studying plant-microbial community interactions under the laboratory conditions.
Keywords: Maize Synthetic community Selective medium Dynamics Community assembly and biological control
Background
In natural settings, plants are associated with myriad microorganisms of extremely high diversity. These microbes exploit the niches provided by plant hosts and form complex microbial communities (Bulgarelli et al., 2012; Ofek-Lalzar et al., 2014; Cardinale et al., 2015; Edwards et al., 2015; Beckers et al., 2016; de Souza et al., 2016; Niu et al., 2017). Such plant-associated microbiomes are able to affect the development and health of the hosts profoundly (Berendsen et al., 2012). Recently, huge amounts of data describing plant microbiome compositions and their dynamics have been obtained by using advanced DNA sequencing technologies and data analysis methods. Much has been learned about the community structure of plant microbiota (Ofek-Lalzar et al., 2014; Bai et al., 2015; Ritpitakphong et al., 2016). However, due to the great complexity, currently it is nearly impossible to directly define experimentally the mechanisms underlying the dynamics of plant microbiome assembly and their beneficial effects on hosts. The establishment of simplified plant-associated microbial communities under controlled laboratory conditions is an approach to overcome the challenges in analyzing the properties of plant microbiota (Bodenhausen et al., 2014; Bai et al., 2015; Lebeis et al., 2015). Testing of hypotheses by targeted manipulation in gnotobiotic systems with simplified synthetic communities become a lot easier (Vorholt et al., 2017).
Previously, through host-mediated selection, we assembled a greatly simplified, yet representative, synthetic bacterial community consisting of seven strains (Stenotrophomonas maltophilia, Ochrobactrum pituitosum, Curtobacterium pusillum, Enterobacter cloacae, Chryseobacterium indologenes, Herbaspirillum frisingense and Pseudomonas putida) (Niu et al., 2017). We found that the removal of E. cloacae caused dramatic changes of the community composition and that this seven-species community protects maize from colonization by a fungal pathogen, Fusarium verticillioides. These results suggest that this synthetic seven-species community has the potential to serve as a useful system to explore how bacterial interspecies interactions affect root microbiome assembly and to dissect the beneficial effects of the root microbiota on hosts under laboratory conditions (Niu et al., 2017). This protocol has been developed to set up a gnotobiotic system for cultivating maize seedlings colonized by the root-associated simplified communities, to track the dynamics of the composition of the simplified communities and to evaluate the in vivo biological control effects of the seven-species community against F. verticillioides.
Materials and Reagents
Consumables
Disposable Petri dishes (VWR, catalog number: 89022-320 )
Pipette tips (Corning, Axygen®, catalog number: T1005WBCRS ; Biotix, catalog number: M-0200-1RCNS )
Parafilm (VWR, catalog number: 52858-000)
Manufacturer: Bemis, catalog number: PM996 .
Inoculation loops (Globe Scientific, catalog number: 130118 )
Centrifuge tubes 2.0 ml (Corning, Axygen®, catalog number: MCT-200-C-S )
Centrifuge tubes 1.5 ml (VWR, catalog number: 20170-038 )
Centrifuge tubes 50 ml (Corning, catalog number: 352098 )
Scalpel blades (Integra LifeSciences, catalog number: 4-110 )
Glass beads (Propper, catalog number: 03000600 )
Paper wipers (KCWW, Kimberly-Clark, catalog number: 34155 )
96-well plates (Corning, catalog number: 351172 )
Cell scrapers (VWR, catalog number: 89260-222 )
Trays (Thermo Fisher Scientific, Nunc, catalog number: 242811 )
Plants
Zea mays cv. Sugar Buns F1 (se+) (Johnny’s Selected Seeds, catalog number: 267 )
Bacterial strains
Stenotrophomonas maltophilia ZK5342, Ochrobactrum pituitosum ZK5343,
Curtobacterium pusillum ZK5344, Enterobacter cloacae ZK5345,
Chryseobacterium indologenes ZK5346, Herbaspirillum frisingense ZK5347 and
Pseudomonas putida ZK5348 (Niu and Kolter, 2017)
These strains can be requested via e-mail: [email protected] or [email protected]
Fungal strain
Fusarium verticillioides MRC826 (Hinton and Bacon, 1995)
Chemical reagents
Ethanol (Decon Labs, catalog number: V1001 )
Bleach (Janitorial Supplies, Clorox®, catalog number: CLO30966CT )
BactoTM Tryptic Soy Broth without Dextrose (BD, catalog number: 286220 )
Soyabean Casein Digest Agar (HiMedia Laboratories, catalog number: GM290-500G )
Agar (BD, catalog number: 214010 )
10x Phosphate buffered saline (PBS) (Lonza, catalog number: 17-517Q )
Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524-50L )
Nalidixic acid (Sigma-Aldrich, catalog number: N8878-5G )
Colistin (Sigma-Aldrich, catalog number: C4461-100MG )
Lincomycin (Sigma-Aldrich, catalog number: 62143-1G )
Chlortetracycline (Sigma-Aldrich, catalog number: C4881-5G )
Erythromycin (Sigma-Aldrich, catalog number: E5389-1G )
Vancomycin (Sigma-Aldrich, catalog number: 75423-5VL )
Sodium chlorite (VWR, catalog number: BDH9286-500G )
Novobiocin (Sigma-Aldrich, catalog number: N1628-1G )
Tobramycin (Sigma-Aldrich, catalog number: T4014-100MG )
Glucose (VWR, catalog number: BDH9230-500G )
Media and buffers (see Recipes)
Tryptone soya agar medium
0.1x Tryptone soya agar medium
Tryptic soy broth medium
1x Phosphate buffered saline (PBS)
½ Murashige and Skoog (MS) agar medium
Selective medium for S. maltophilia ZK5342
Selective medium for O. pituitosum ZK5343
Selective medium for C. pusillum ZK5344
Selective medium for E. cloacae ZK5345
Selective medium for C. indologenes ZK5346
Selective medium for H. frisingense ZK5347
Selective medium for P. putida ZK5348
Potato dextrose agar medium
Water agar medium
Equipment
Forceps
Pipettes (Gilson, models: P20, P200 and P1000, catalog numbers: F123600 , F123601 and F123602 ; Thermo Fisher Scientific, model: F1-ClipTipTM, catalog numbers: 4661140N and 4661130N )
Class II biological safety cabinet (Thermo Fisher Scientific, model: HerasafeTM KS9 )
Centrifuge (Eppendorf, model: 5424 )
Vortex (Scientific Industries, model: Vortex-Genie 2, catalog number: G560 )
Spectrophotometer (Beckman Coulter, model: DU 640 )
Sonicator (Qsonica, model: Q125 , catalog number: Q125-110)
Balance (Mettler-Toledo International, catalog number: AG135 )
Hemacytometer (Hausser Scientific, catalog number: 1492 )
Microscope (ZEISS, model: Axioscop 2 plus )
Stereoscope (ZEISS, model: Stemi SV 6 )
Software
RStudio (version 0.99.903)
QIIME (version 1.6.0)
PRISM (version 6.0c)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：牛, 犇. and Kolter, R. (2018). Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect. Bio-protocol 8(12): e2885. DOI: 10.21769/BioProtoc.2885.
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Category
Microbiology > Microbe-host interactions > In vivo model
Microbiology > Community analysis > Gnotobiotic system
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2,886 | https://bio-protocol.org/exchange/protocoldetail?id=2886&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation of Microvascular Endothelial Cells
KC Kenneth C.P. Cheung
FM Federica M. Marelli-Berg
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2886 Views: 14266
Edited by: Ivan Zanoni
Reviewed by: Lokesh KalekarSalma Merchant
Original Research Article:
The authors used this protocol in Nov 2017
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Original research article
The authors used this protocol in:
Nov 2017
Abstract
The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. Murine endothelial cell culture is an important tool for cardiovascular disease research. This protocol demonstrates a quick, efficient method for the isolation of microvascular endothelial cells from murine tissues without any special equipment. To isolate endothelial cells, the lung or heart were mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained was then incubated with an anti-CD31, anti-CD105 antibody and with biotinylated isolectin B-4. The endothelial cells were harvested using magnetic bead separation with rat anti-mouse Ig- and streptavidin-conjugated microbeads. Endothelial cells were expanded and collected for subsequent analyses. The morphological and phenotypic features of these cultures remained stable over 10 passages in culture. There was no overgrowth of contaminating cells of non-endothelial origin at any stage.
Keywords: Primary culture Endothelial cells Tight junctions CD31 Pecam1
Background
Microvascular endothelial cells play a central role in the development of immune responses by regulating leukocyte recirculation and as antigen presenting cells to T lymphocytes. The wellbeing of the endothelium is essential to vascular homeostasis. The dysfunctional endothelium participates in various cardiovascular disorders, including atherosclerosis, vasculitis and ischemia/reperfusion injuries (Cid et al., 2004; Wang et al., 2007). Therefore, in vitro endothelial cell cultures are important tools for studying vascular physiology and disease pathology. However, the isolation of primary murine endothelial cells is considered particularly difficult because most protocols described have involved the perfusion of organs or large vessels with digesting enzymes and time-consuming purification process (Gumkowski et al., 1987).
The purpose of this protocol is to provide a simple method to isolate and expand endothelial cells from the lung/heart without using any special equipment. Using this method, we previously complemented in vivo studies demonstrating the importance of CD31 signaling in endothelial cells cytoprotection (Cheung et al., 2015).
Materials and Reagents
Materials
Pipette tips
Multiwell plate (cell culture grade) (Greiner Bio One International, catalog number: 662160 )
50 ml centrifuge tubes (cell culture grade) (Greiner Bio One International, catalog number: 210261 )
10 ml disposable pipette (Greiner Bio One International, catalog number: 607160 )
Cell strainers (100 µm, Corning, catalog number: 352360 ; 70 µm, Corning, catalog number: 352350 )
Scalpel
miniMACS separation unit (Miltenyi Biotec, catalog number: 130-042-102 )
Note: Magnetic cell sorting of labeled EC was performed using a miniMACS separation unit (Miltenyi Biotec, Bisley, Surrey, UK) including two magnets. Labeled cells were incubated with MACS magnetic goat anti-rat IgG (H+L) (Miltenyi Biotec) MicroBeads and streptavidin (Miltenyi Biotec) MicroBeads and then separated using a high gradient magnetic separation column (MS+ columns, Miltenyi Biotec) placed on the separation unit, according to the manufacturer’s instructions.
High gradient magnetic separation column (MS+ columns) (Miltenyi Biotec, catalog number: 130-042-201 )
Animals
Mice (Balb/c, age 6 weeks up to 1 year from Charles River, UK or the in-house breeding facility)
Reagents
Ice
Isoflurane
Phosphate buffered saline solution (PBS, Gibco)
Collagenase type II (Thermo Fisher Scientific, GibcoTM, catalog number: 17101015 )
EC media
DNaseI solution
0.125% trypsin in 0.2% EDTA (Life Technologies)
Dako mounting media (Dako)
MicroBeads and streptavidin (Miltenyi Biotec, catalog number: 130-048-101 )
Antibodies
Biotinylated isolectin B4 (purchased from Vector Laboratories, Peterborough, UK)
Note: The anti-CD40 mAb 3/23 (rat IgG2a) (Van Den Berg et al., 1996) was a kind gift from Dr. G. Klaus (National Institute for Medical Research, London, UK).
Rat IgG2a (clone R35-95, BD, PharmingenTM, catalog number: 553927 )
Hamster Igs (BD, CompBeadTM, catalog number: 552845 )
Mouse IgG1 (TdT Cocktail Control, Harlan Sera-Lab, Oxon, UK, Thermo Fisher Scientific, catalog number: 31903 )
Note: The above mAbs 16b and 16c were used as isotype-matched control antibodies in staining experiments: rat IgG2a (clone R35-95); hamster Igs. To block Fc receptors, mouse IgG2a and mouse IgG1 (TdT Cocktail Control, Harlan Sera-Lab, Oxon, UK) were used.
MACS magnetic goat anti-rat IgG (H+L) (Miltenyi Biotec, catalog number: 130-048-501 )
Rat IgG2b anti-mouse CD16/CD32 monoclonal antibody (BD, catalog number: 553141 )
Secondary antibody conjugated rhodamine red-X (Molecular Probes)
FACS
Note: The following antibodies were purchased from Pharmingen (La Jolla, CA).
CD31 (PECAM-1, clone MEC 13.3, rat IgG2a, k) (BD, PharmingenTM, catalog number: 550274 )
CD105 (Endoglin, clone MJ7/18, rat IgG2a) (BD, PharmingenTM, catalog number: 550546 )
Immuno Fluorescence Staining
PECAM-1(MEC 13.3) (Santa Cruz Biotechnology, catalog number: sc-18916 )
Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, GibcoTM, catalog number: 41966-052 )
Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030 )
10,000 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360039 )
HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630056 )
1% non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
2-mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
Heat-inactivated fetal calf serum (FCS; Globepharm, Esher, UK)
EC growth supplement (Sigma-Aldrich, catalog number: E0760 )
2% gelatin (type B from bovine skin, Sigma-Aldrich, catalog number: G7765 ) coated tissue culture flasks (Nunc, Life Technologies, Paisley, UK)
Working medium (see Recipes)
Equipment
Pipettes
Sterile beakers 100-150 ml (sterilize at 180 °C)
Laminar flow work bench
Tweezers (sterilize at 180 °C)
Scissors (sterilize at 180 °C)
Shaker
Water bath
Centrifuge (Hettich Instruments, model: UNIVERSAL 320 R )
Fixed-angle rotor (Hettich Instruments, catalog number: 1620A )
EPICS Profile Cytometer (Coulter Electronics, Luton, UK)
Fluorescence microscopy (Zeiss epi-fluorescent microscope)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Cheung, K. C. and Marelli-Berg, F. M. (2018). Isolation of Microvascular Endothelial Cells. Bio-protocol 8(12): e2886. DOI: 10.21769/BioProtoc.2886.
Download Citation in RIS Format
Category
Cancer Biology > Inflammation > Biochemical assays
Cell Biology > Cell isolation and culture > Cell isolation
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2,887 | https://bio-protocol.org/exchange/protocoldetail?id=2887&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Procedure for Precise Determination of Glutathione Produced by Saccharomyces cerevisiae
JK Jyumpei Kobayashi
DS Daisuke Sasaki
Akihiko Kondo
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2887 Views: 7092
Edited by: Valentine V Trotter
Reviewed by: Pushpendra Singh
Original Research Article:
The authors used this protocol in Oct 2013
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The authors used this protocol in:
Oct 2013
Abstract
In bioproduction, yields of products must be calculated precisely for accurate evaluation of various fermentation conditions. To evaluate productivity of microorganisms, product amounts per unit of medium volume (e.g., mg-product/L-broth), and/or product amounts per unit of a microorganism amount (e.g., mg-product/mg-dry cell weight) are often used. Nonetheless, detailed procedures for calculation of these production yields are often omitted in research articles, whereas methods for product quantification are described well. Here, we describe a detailed calculation procedure from our previous studies on glutathione production by Saccharomyces cerevisiae. This procedure can be applied to various other products and microorganisms, and therefore, may prove to be useful in various other bioproduction studies.
Keywords: Saccharomyces cerevisiae Glutathione GSH GSSG Aggregated microorganisms
Background
Glutathione is the most abundant thiol-containing tripeptide in all living organisms and functions as a bioactive substance with varied roles in cells, e.g., as redox and antidotal agents. Therefore, glutathione is widely used in the medical, food, and cosmetic industries nowadays, and the demand has increased in recent years. Glutathione is industrially produced mainly by fermentation using Saccharomyces cerevisiae, which originally contains a high concentration of glutathione and has served as a safe, food-producing microorganism. Studies of microbial glutathione production in various microorganisms will become more important in the future. To evaluate the productivity in terms of glutathione via fermentation by various microorganisms, here we describe our detailed procedures of sample preparation, quantification of reduced and oxidized glutathione by high performance chromatography (HPLC), and calculations of two types of yield (Hara et al., 2012; Hara et al., 2015; Kiriyama et al., 2013; Kobayashi et al., 2017).
Materials and Reagents
200 μl pipette tips (FCR&Bio, catalog number: AG-200-FP-Y )
1,000 μl pipette tips (FCR&Bio, catalog number: AG-1000B )
1.5 ml microcentrifuge tubes (FUKAEKASEI and WATSON, catalog number: 131-815C )
50 ml centrifuge tubes (Corning, catalog number: 430291 )
Filter unit with 0.22 μm pore size (Shimadzu, catalog number: GLCTD-MCE1322 )
Saccharomyces cerevisiae
Yeast cell culture medium
Ultrapure water (Milli-Q)
Ice
Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate (NACALAI TESQUE, catalog number: 15111-45 )
Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 163-04265 )
1-Heptanesulfonate (NACALAI TESQUE, catalog number: 31528-92 )
Phosphoric acid (Wako Pure Chemical Industries, catalog number: 162-20492 )
Methanol (NACALAI TESQUE, catalog number: 21929-23 )
Yeast extract dried (NACALAI TESQUE, catalog number: 15838-45 )
Hipolypepton (NIHON PHARMACEUTICAL, catalog number: 392-02115 )
D-(+)-Glucose (NACALAI TESQUE, catalog number: 16805-35 )
Aureobasidin A (Takara Bio, catalog number: 630499 )
99.5% Ethanol (NACALAI TESQUE, catalog number: 14713-95 )
Agar powder (NACALAI TESQUE, catalog number: 01028-85 )
Standard Buffer Solution (pH 4.01) (NACALAI TESQUE, catalog number: 37219-75 )
Standard Buffer Solution (pH 6.86) (NACALAI TESQUE, catalog number: 37220-35 )
Standard Buffer Solution (pH 9.18) (NACALAI TESQUE, catalog number: 37221-25 )
YPD medium (see Recipes)
Aureobasidin A stock solution (see Recipes)
500 mM EDTA (see Recipes)
100 mM EDTA (see Recipes)
HPLC mobile phase (see Recipes)
Equipment
Micropipetter (volume range 20-200 μl) (Nichiryo, model: NPX-200 )
Micropipetter (volume range 100-1,000 μl) (Nichiryo, model: NPX-1000 )
Block heater (ASTEC, model: BI-516C )
Aluminum microtube rack (Bio Medical Science, model: BMS-D081 )
Centrifuge (KUBOTA, model: 3740 )
Centrifuge rotor for 1.5 ml microcentrifuge tubes (maximum capacity is 21,880 x g) (KUBOTA, model: AF-2236 )
Centrifuge rotor for 50 ml centrifuge tubes (maximum capacity is 22,140 x g) (KUBOTA, model: AF-5004CH )
Glass test tube (NICHIDEN RIKA GLASS, model: 101015 )
Baffled Erlenmeyer flask (AGC TECHNO GLASS, model: 4551FK200R )
Silicone sponge tapered plug closure (Shin-Etsu Polymer, model: T-19 )
Silicone sponge closure (Shin-Etsu Polymer, model: C-40 )
Spectrophotometer (Shimadzu, model: UVmini-1240 )
Spectrophotometer cell (Hellma, model: 104-10-40 )
High-performance liquid chromatography system (Shimadzu, model: Prominence)
Octa decyl silyl (ODS) column (YMC, model: YMC-Pack ODS-A )
Electronic scale (Mettler-Toledo International, model: XS105DU )
Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
Ice-making machine (HOSHIZAKI, model: CM-100K )
Deep freezer (temperature range: from -20 to -30 °C) (Panasonic, model: MDF-U539 )
Deep freezer (temperature range: from -50 to -85 °C) (PHC, model: MDF-U33V-PJ )
pH meter (Horiba, model: F-52 )
Forced air flow oven (Tokyo Rikakikai, model: WFO-1210 )
Disposable loop (AS ONE, model: 6-488-02 )
Incubator for solid preculture (Sanyo, model: MIR-153 )
Incubator for liquid preculture (TAITEC, model: BR-43FL・MR )
Incubator for glutathione production (TAITEC, model: G・BR-200 )
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kobayashi, J., Sasaki, D. and Kondo, A. (2018). A Procedure for Precise Determination of Glutathione Produced by Saccharomyces cerevisiae. Bio-protocol 8(12): e2887. DOI: 10.21769/BioProtoc.2887.
Download Citation in RIS Format
Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Quantification
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2,888 | https://bio-protocol.org/exchange/protocoldetail?id=2888&type=0 | # Bio-Protocol Content
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Protocols to Study Declarative Memory Formation in Mice and Humans: Optogenetics and Translational Behavioral Approaches
AS Azza Sellami*
AA Alice Shaam Al Abed*
LB Laurent Brayda-Bruno*
NE Nicole Etchamendy*
SV Stéphane Valério*
MO Marie Oulé
LP Laura Pantaléon
VL Valérie Lamothe
MP Mylène Potier
KB Katy Bernard
MJ Maritza Jabourian
CH Cyril Herry
NM Nicole Mons
AM Aline Marighetto
*Contributed equally to this work
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2888 Views: 5892
Edited by: Edgar Soria-Gomez
Reviewed by: Francesco Papaleo
Original Research Article:
The authors used this protocol in Sep 2017
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Sep 2017
Abstract
Declarative memory formation depends on the hippocampus and declines in aging. Two functions of the hippocampus, temporal binding and relational organization (Rawlins and Tsaltas, 1983; Eichenbaum et al., 1992; Cohen et al., 1997), are known to decline in aging (Leal and Yassa, 2015). However, in the literature distinct procedures have been used to study these two functions. Here, we describe the experimental procedures used to investigate how these two processes are related in the formation of declarative memory and how they are compromised in aging (Sellami et al., 2017). First, we studied temporal binding using a one-trial learning procedure: trace fear conditioning. It is classical Pavlovian conditioning requiring temporal binding since a brief temporal gap separates the conditioned stimulus (CS) and unconditioned stimulus (US) presentations. We combined the trace fear condition procedure with an optogenetic approach, and we showed that the temporal binding relies on dorsal (d)CA1 activity over temporal gaps. Then, we studied the interaction between temporal binding and relational organization in declarative memory formation using a two-phase radial-maze task in mice and its virtual analog in humans. The behavioral procedure comprises an initial learning phase where subjects learned the constant rewarding /no rewarding valence of each arm, followed by a test phase where the reward contingencies among the arms remained unchanged but where the arms were recombined to assess flexibility, a cardinal property of declarative memory. We demonstrated that dCA1-dependent temporal binding is necessary for the development of a relational organization of memories that allows flexible declarative memory expression. Furthermore, in aging, the degradation of declarative memory is due to a reduction of temporal binding capacity that prevents relation organization.
Keywords: Trace fear conditioning Radial maze Virtual radial maze Optogenetics Channelrhodopsin Archeorhodopsin
Background
Declarative memory formation (i.e., memory of everyday facts and events) is dependent on the hippocampus and declines in aging (Leal and Yassa, 2015). Two fundamental functions of the hippocampus are known to be age-sensitive. First, the hippocampus supports ‘temporal binding’, (Rawlins and Tsaltas, 1983), a function that allows association in memory of discontiguous events. Trace conditioning tasks allow the demonstration of the critical role of hippocampus in temporal binding (Solomon et al., 1986; Clark and Squire, 1998; LaBar and Disterhoft, 1998; Huerta et al., 2000) and its decline in aging (Disterhoft and Oh, 2007). Second, the hippocampus is critical to the formation of ‘relational organization’, a function that links memorized information/events and consequently supports cardinal flexibility of declarative memory, exemplified in the capability to make inferences from memory (Bunsey and Eichenbaum, 1996) or to compare separately acquired information to guide a choice decision in a novel situation (Etchamendy et al., 2003; Mingaud et al., 2007). This capability is compromised in aging (Rapp et al., 1996; Marighetto et al., 1999; Etchamendy et al., 2001; Mingaud et al., 2008). However, temporal binding and relational organization have always been studied separately. Here, we describe procedures to study how these two processes are related. Our previous study showed that these two hippocampal functions are causally related in the formation of declarative memory by using a specific experimental protocol combining behavioral and optogenetics approaches (Sellami et al., 2017). Specifically, temporal binding is a necessary condition for the relational organization of discontiguous events (Figure 1). We found out that the formation of a relational memory is limited by the capability of temporal binding, which depends on dorsal (d)CA1 activity over time intervals and diminishes in aging. Conversely, relational representation is successful, even in aged individuals, when the demand on temporal binding is minimized, showing that the relational/declarative memory per se is not impaired in aging. Thus, bridging temporal intervals by dCA1 activity is a critical foundation of relational representation, and a deterioration of this mechanism is responsible for the age-associated memory impairment.
Figure 1. Temporal binding is critical to associate discontiguous events into a relational representation allowing flexible expression of memory
Part I: Virus transfection and fiber implantation
Materials and Reagents
Glass capillaries (WPI, catalog number: 1B150F-4 )
Silicone catheter (Dominique DUTSCHER, catalog number: 351070 )
Syringe 1 ml (Henke-Sass, Wolf, catalog number: 4010.200V0 )
Syringe 5 ml (Henke-Sass, Wolf, catalog number: 4050.000V0 )
Petri dish 100 x 15 mm
Implantable Fiber Optic Cannulae (Thorlabs, catalog number: CFMLC12L02 )
Optic fiber (Thorlabs, catalog number: FT200EMT )
Patch cable (Thorlabs, catalog number: M83L01 )
Ceramic split mating sleeve (Thorlabs, catalog number: ADAL1 )
Parafilm (Heathrow Scientific, Bemis, catalog number: HEA234526A )
Surgical blades
Screws (MicroFastenings, catalog number: M0.6x1.5 )
Needle 26 G x ½" (Terumo Medical, catalog number: NN-2613R )
Needle 23 G x 1¼" (Henke-Sass, Wolf, catalog number: 4710006030 )
Cotton swab (The Lab Depot, catalog number: 394305 )
Stitching kit and sutures (Péters Surgical, catalog number: 87001F )
6-well culture plate (Corning, Falcon®, catalog number: 353046 )
Microscope slide (Thermo Fisher Scientific, catalog number: LCSF02 )
Cover slips (Knittel Glass, catalog number: VD12450Y1A.01 )
Brush (Henry Schein France, catalog number: 878-7825 )
1 L Solvent Bottle (Thermo Fisher Scientific, catalog number: 045900 )
Whatman® paper filter (GE Healthcare, catalog number: 1213125 )
Young adult (3- to 4-mo-old) and aged (21-to 23-mo-old) C57BL/6 male mice (Charles River)
AAV vectors:
AAV-CAMKIIa-hChR2(H134R)-EYFP (University of North Carolina (UNC) Vector Core)
AAV-CAMKIIa-ArchT-GFP (University of North Carolina (UNC) Vector Core)
AAV-CAMKIIa-GFP (University of North Carolina (UNC) Vector Core)
Isoflurane 1,000 mg/g (4% induction and 1-2% for maintenance, Iso-Vet)
Betadine
Super glue (Loctite)
Liquid fix glue: Methylmethacrylate (Sigma-Aldrich, catalog number: M55909-25ML )
Super-Bond C&B dental cement (Sun Medical, catalog number: P021E/0A )
Metacam: Méloxicam 1.5 mg/ml analgesic (Boehringer Ingelheim)
Lurocaine: Lidocaïne 20 mg (Vetoquinol)
70% ethanol solution
Lacrigel: eye ointment (Europhta)
Sulmidol: Sulfapyridine 100 mg (MSD, santé animale)
Hydrogen peroxide (Sigma-Aldrich, catalog number: H1009 )
Sodium phosphate dibasic, Na2HPO4 (Sigma-Aldrich, catalog number: S0876 )
Sodium phosphate monobasic, NaH2PO4 (Sigma-Aldrich, catalog number: S0751 )
Sodium chloride, NaCl (Sigma-Aldrich, catalog number: 433209 )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 441244 )
Sodium Hydroxide solution 1.0 N, NaOH (Sigma-Aldrich, catalog number: S2770 )
FluorSave reagent (Merck, catalog number: 345789 )
0.1 M PBS (see Recipes)
0.2 M PB (see Recipes)
PFA 4%/0.1 M PB solution (see Recipes)
Equipment
Small scissors (World Precision Instruments, catalog number: 504615 )
Bone scraper (World Precision Instruments, catalog number: 503759 )
Drill (RWD Life Science, catalog number: 78001 )
Fine tip forceps (World Precision Instruments, catalog number: 501975 )
Needle holder with Suture Scissors (World Precision Instruments, catalog number: 500023 )
Screwdriver (World Precision Instruments, catalog number: 501635 )
Compact Power and Energy Meter Console (Thorlabs, catalog number: PM100D )
Anesthesia system for isoflurane (Datex Ohmeda ISO Isoflurane Anesthesia Vaporizer Tec 7)
Pipette puller (Sutter Instrument, model: P97 )
Picospritzer (Parker Hannifin)
Hair clipper (PHYMEP, model: Contura Shaver )
Heating pad (Tem Sega, model: THERM250 )
Mouse stereotaxic apparatus (KOPF INSTRUMENTS, model: 942 )
Binocular loupe (Leica Microsystems, model: Leica S6E )
Perfusion pump (Cole-Parmer, model: Master Flex® L/S® )
Vibratome (Leica Biosystems, model: Leica VT 1000S )
Microscope (ZEISS, model: Axio Imager A2 )
The Mouse Brain in Stereotaxic Coordinates, 2001
TTL pulse generator (Imetronic, Pessac, France)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sellami, A., Al Abed, A. S., Brayda-Bruno, L., Etchamendy, N., Valério, S., Oulé, M., Pantaléon, L., Lamothe, V., Potier, M., Bernard, K., Jabourian, M., Herry, C., Mons, N. and Marighetto, A. (2018). Protocols to Study Declarative Memory Formation in Mice and Humans: Optogenetics and Translational Behavioral Approaches. Bio-protocol 8(12): e2888. DOI: 10.21769/BioProtoc.2888.
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Category
Neuroscience > Behavioral neuroscience > Learning and memory
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2,889 | https://bio-protocol.org/exchange/protocoldetail?id=2889&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803
CG Chiara Gandini
Søren Husted
SS Sidsel Birkelund Schmidt
Published: Vol 8, Iss 12, Jun 20, 2018
DOI: 10.21769/BioProtoc.2889 Views: 5055
Reviewed by: Nidhi Sharma
Original Research Article:
The authors used this protocol in Jul 2017
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Original research article
The authors used this protocol in:
Jul 2017
Abstract
Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and metal binding in the size separated complexes is analyzed by hyphenation with ICP-QQQ-MS.
Keywords: Cyanobacteria Synechocystis Manganese Iron Magnesium Thylakoid membranes Speciation analysis ICP-MS
Background
The process of oxygenic photosynthesis requires metals due to their essential functions as cofactors and catalysts in the photosynthetic electron transport chain. The photosynthetic apparatus requires iron (Fe) in the form of either Fe-S clusters, heme-bridged Fe and non-heme Fe, copper (Cu) in the soluble mobile electron carrier protein plastocyanin, magnesium (Mg) in chlorophylls, calcium (Ca) and manganese (Mn) in the oxygen evolving complex of photosystem II (PSII). Tight control of metal allocation inside photosynthetic cells is essential for cell survival since an imbalanced metal accumulation induces mismetallation and inactivation of the various metallo-enzymes. An accurate analysis of the concentration and allocation of metals in photosynthetic cells is therefore important to investigate the role of key factors and proteins involved in metal homeostasis. While the method described in Brandenburg et al. (2017) accurately quantifies the periplasmic and intracellular pools of Mn in Synechocystis cells, the protocol presented here provides a wider overview, quantifying metals in whole Synechocystis cells, isolated thylakoids, and the bioactive metal pool in fractionated photosynthetic complexes. This protocol is partially based on the method described in Schmidt et al. (2015) for barley thylakoids.
Materials and Reagents
Pipette tips
50 ml conical tubes (Greiner Bio One International, catalog number: 227261 )
1.5 and 2 ml Eppendorf tubes (Eppendorf, catalog numbers: 0030120086 and 0030120094 , respectively)
Microwave Teflon tubes, 8 ml (VWR, catalog number: 525-0178 )
Nylon membrane filters, 0.45 µm (Frisenette, catalog number: 13NY045-100 )
Syringe, 1 ml (Frisenette, catalog number: 9161406 )
NanoVipers fingertight fittings, 150 µm (Thermo Fisher Scientific, catalog number: 6041.5820 )
Synechocystis sp. PCC6803 GT (glucose tolerant strain, from Dr. Himadri Pakrasi laboratory, Department of Biology, Washington University, St Louis, MO, USA)
Multi-metal calibration standards for ICP-MS calibration (CPI International, catalog numbers: P/N4400-132565A and P/N4400-132565B )
EDTA, BioUltra (Sigma-Aldrich, catalog number: E1644-100G )
Tricine (Sigma-Aldrich, catalog number: T9784-25G )
Lysozyme (Sigma-Aldrich, catalog number: 62971-50G-F )
Sucrose, BioExtra (Sigma-Aldrich, catalog number: S7903-1KG )
Sodium chloride (NaCl), BioExtra (Sigma-Aldrich, catalog number: S7653-250G )
Magnesium chloride (MgCl2·7H2O), BioExtra (Sigma-Aldrich, catalog number: M2670-1KG )
Na-ascorbate, BioExtra (Sigma-Aldrich, catalog number: 11140-250G )
Sodium fluoride (NaF), BioExtra (Sigma-Aldrich, catalog number: S7920-100G )
Liquid nitrogen
PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225 )
Glass beads (Sigma-Aldrich, catalog number: G4649 )
Milli-Q water
PlasmaPURE 67-69% HNO3 (SCP SCIENCE, catalog number: 250-039-175 )
30% H2O2 (prepare 15% H2O2 with Milli-Q water) (Sigma-Aldrich, catalog number: 31642 )
n-Dodecyl-β-D-Maltopyranoside (β-DM) (Anatrace, catalog number: D310LA )
Bis-Tris, BioExtra (Sigma-Aldrich, catalog number: B7535-100G )
Glycerol, BioExtra (Sigma-Aldrich, catalog number: G6279 )
Betaine, BioUltra (Sigma-Aldrich, catalog number: 61962-250G )
Pefabloc SC (Roche Diagnostics, catalog number: 11429868001 )
Apple leaves, standard reference material (Sigma-Aldrich, catalog number: NIST1515 )
Glucose (Sigma-Aldrich, catalog number: G8270-1KG )
Na2S2O3·5H2O (Merck, catalog number: 1.06516.0500 )
FeNH4 citrate (MP Biomedicals, catalog number: 02158040 )
Na2CO3 (Sigma-Aldrich, catalog number: S7795-500G )
K2HPO4 (Sigma-Aldrich, catalog number: P8281 )
NaNO3 (Sigma-Aldrich, catalog number: S8170-250 )
MgSO4 (Sigma-Aldrich, catalog number: M2643-500G )
CaCl2 (Sigma-Aldrich, catalog number: C5670-500G )
Citric acid (Sigma-Aldrich, catalog number: 251275-100G )
Na2-EDTA (Sigma-Aldrich, catalog number: E4884-500G )
H3BO3 (Sigma-Aldrich, catalog number: B6768-500G )
MnCl2 (Sigma-Aldrich, catalog number: 244589-50G )
ZnSO4 (Sigma-Aldrich, catalog number: Z0251-100G )
NaMoO4 (Sigma-Aldrich, catalog number: M1003-100G )
CuSO4 (Sigma-Aldrich, catalog number: 209198-100G )
Co(NO3)2 (Alfa Aesar, catalog number: 36418.22 )
BG11-G (see Recipes)
100x BG-FPC (see Recipes)
1,000x trace element stock (see Recipes)
FeNH4 citrate stock (see Recipes)
Na2CO3 stock (see Recipes)
K2HPO4 stock (see Recipes)
EDTA washing buffer (see Recipes)
Tricine buffer (see Recipes)
Lysozyme solution (see Recipes)
Homogenization buffer (see Recipes)
Tricine-NaF buffer (see Recipes)
Storage buffer (see Recipes)
Solubilization buffer (see Recipes)
Detergent solution (see Recipes)
Mobile phase buffer (see Recipes)
Equipment
Pipettes
Erlenmeyer flasks, 2 L (Fisher Scientific, catalog number: 11961566)
Manufacturer: Pyrex, catalog number: 1130/30D .
Orbital shaker (Eppendorf, New BrunswickTM, model: Innova® 2150, catalog number: M1194-0010 )
Eppendorf centrifuge 5418 R, refrigerated (Eppendorf, model: 5418 R, catalog number: 5401000013 )
Eppendorf centrifuge 5430 R, refrigerated (Eppendorf, model: 5430 R, catalog number 5428000210 )
Beckman Coulter centrifuge (Beckman Coulter, model: Avanti® J-25 ) with type 19 rotor, fixed angle
250 ml bottles (Beckman Coulter, catalog number: 325620 )
Microfluidizer Processor (Microfluidics, model: M-110L )
Light microscope (ZEISS, model: Axiovert 135 TV )
Bead mill TissueLyser II (QIAGEN, catalog number: 85300 )
TissueLyser II adapter set (QIAGEN, catalog number: 69982 )
Heraeus Fresco 21 Microcentrifuge, refrigerated (Thermo Fisher Scientific, catalog number: 75002425 )
HPLC Microvials PP, 300 µl and screw cap PP, PFTE (VWR, catalog numbers: 548-0440 and 548-0787 )
Size exclusion column, Biobasic SEC-1000 analytical column (300 x 7.8 mm) and guard column (30 x 7.8 mm), 5 µm particle size, 1000Å pore size (Thermo Fisher Scientific, catalog number: 73605-307846 and 73605-037821 )
Pressurized Ultrawave System (Ultrawave system, Milestone Srl, Sorisole, Italy)
HPLC system, bio-inert (i.e., the mobile phase and samples have no contact with metal parts) (Thermo Fisher Scientific, Dionex, model: UltiMate 3000 )
DionexTM ICS-5000+ DP Dual Pumps (Thermo Fisher Scientific, model: ICS-5000+ DP )
ICP-QQQ-MS (Agilent Technologies, model: 8800 Triple Quadrupole ICP-MS )
Nebulizer, Ari Mist HP (Burgener Research, catalog number: AM HP 5500 )
Gases for ICP: Liquid Argon, grade 5.0, and reaction gas: 20% Oxygen in Argon (AGA, catalog numbers: 107407 and 714039 )
Software
MassHunter 4.2 Workstation software 8800 ICP-QQQ data analysis (Agilent Technologies)
Procedure
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Gandini, C., Husted, S. and Schmidt, S. B. (2018). Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803. Bio-protocol 8(12): e2889. DOI: 10.21769/BioProtoc.2889.
Download Citation in RIS Format
Category
Plant Science > Plant biochemistry > Other compound
Plant Science > Plant physiology > Photosynthesis
Biochemistry > Other compound > Elements
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