Flow Cytometry Purification of Mouse Meiotic Cells

Summary

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.

Abstract

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 cells^1-5. 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 staining^7,8. 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 expression^9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination¹¹.

Protocol

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.

1. Testis Dissociation

1. Place one decapsulated testis in a 15-ml tube.
2. Add 3-ml of Gey's Balance Salt Solution (GBS....

Discussion

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 extraction^9,10, 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase Type-1	Worthington Biochemical	CLSS1	Dissolve to 12,000U/ml in GBSS, store at 4°C
Trypsin	Worthington Biochemical	TRL3	Dissolve in 1mM HCl to 50mg/ml, store at 4°C
H–chst 33342	Acros Organics	230001000	Saturated solution (10mg/ml in DMSO, store at 4°C
DNAse I	Sigma-Aldrich	DNEP	Dissolve in water/50% glycerol to 1mg/ml, store at -20°C
Gey’s Balance Salt Solution	Sigma-Aldrich	G9779
6" Transfer Pipet	Fisher Scientific	137119D