Name: flow cytometry purification of mouse meiotic cells
Uploaded: 2011-04-15T11:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry Purification of Mouse Meiotic Cells at JoVE.com

This project was supported in part by monies from the State of Florida to Scripps and award numbers R01GM085079 and R21HD061304 from the National Institute of General Medical Sciences and the National Institute of Child Health and Human Development, respectively. This is manuscript number 20917 of The Scripps Research Institute.

This protocol can be separated in two major steps: (1) the dissociation and Hoechst 33342 staining of mouse testis cells followed by, if necessary, (2) FACS sorting of the relevant meiotic fractions, including all stages of meiosis, from germ cells to round spermatids. Once collected, these highly enriched meiotic populations can be used for a wide range of analysis. This protocol describes the dissociation of one adult testis; volumes can be adapted accordingly for juveniles or for additional testes.
<p class="jove...

<ol>
	<li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Grabske, R. J., Lake, S., Gledhill, B. L., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+elutriation:+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity.">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell. Physiol. 86, 177-189 (1975).</li><li>Purification, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=culture,+and+fractionation+of+spermatogenic+cells.">culture, and fractionation of spermatogenic cells.</a> Methods Enzymol. 225, 84-113 (1993).</li><li>Romrell, L. J., Bellve, A. R., Fawcett, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.+A+morphological+characterization.">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li><li>Meistrich, M. L., Trostle, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients.">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li><li>Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr. Biol. 16, 660-667 (2006).</li><li>Lassalle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+Population'+cells+in+adult+mouse+testis+express+Bcrp1+gene+and+are+enriched+in+spermatogonia+and+germinal+stem+cells.">Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells.</a> Development. 131, 479-487 (2004).</li><li>Bastos, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</li><li>Chowdhury, R., Bois, P. R., Feingold, E., Sherman, S. L., Cheung, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+variation+in+human+meiotic+recombination.">Genetic analysis of variation in human meiotic recombination.</a> PLoS Genet. 5, e1000648-e1000648 (2009).</li><li>Roig, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+TRIP13/PCH2+is+required+for+recombination+and+normal+higher-order+chromosome+structure+during+meiosis.">Mouse TRIP13/PCH2 is required for recombination and normal higher-order chromosome structure during meiosis.</a> PLoS Genet. 6, (2010).</li><li>Getun, I. V., Wu, Z. K., Khalil, A. M., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+occupancy+landscape+and+dynamics+at+mouse+recombination+hotspots.">Nucleosome occupancy landscape and dynamics at mouse recombination hotspots.</a> EMBO Rep. 11, 555-560 (2010).</li><li>Romanienko, P. J., Camerini-Otero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Spo11+gene+is+required+for+meiotic+chromosome+synapsis.">The mouse Spo11 gene is required for meiotic chromosome synapsis.</a> Mol. Cell. 6, 975-987 (2000).</li></ol>

The protocol presented herein allows one to simultaneously purify from adult male mice the entire range of the meiotic stage cells with very high purity, allowing investigators to study the dynamics of this fundamental process. Purified cells can be used for numerous applications ranging from RNA extraction9,10, nucleosome mapping11, recombinant molecule detection, protein analyses, and many more. However, detection methods have to be adapted to the amount of meiotic cells purified. Moreover, with t...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Collagenase Type-1</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>CLSS1</td>
<td>Dissolve to 12,000U/ml in GBSS, store at 4&deg;C</td>
</tr>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>TRL3</td>
<td>Dissolve in 1mM HCl to 50mg/ml, store at 4&deg;C</td>
</tr>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Arcos</td>
<td>230001000</td>
<td>Saturated solution (10mg/ml in DMSO, store at 4&deg;C</td>
</tr>
<tr>
<td>DNAse I</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>DNEP</td>
<td>Dissolve in water/50% glycerol to 1mg/ml, store at -20&deg;C</td>
</tr>
<tr>
<td>Gey&#x2019;s Balance Salt Solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G9779</td>
<td>&nbsp;</td>
<tr>
<td>6" Transfer Pipet</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>137119D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

flow cytometry purification of mouse meiotic cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

Watch this Scientific Journal Video about Flow Cytometry Purification of Mouse Meiotic Cells at JoVE.com

Flow Cytometry Purification of Mouse Meiotic Cells

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Meiosis

SCIENTISTS IN ACTION

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...