Flow Cytometry of Murine Spermatocytes

Abstract

Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back‐gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis‐defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis. © 2015 by John Wiley & Sons, Inc.

Multispecies Purification of Testicular Germ Cells

“…Since the first reports over a decade ago, flow cytometry of Hoechst-stained murine male germ cells has been slowly revisited and optimized to isolate premeiotic (Spg), meiotic (preleptotene, leptotene, zygotene, pachytene, diplotene, and Spc II) and postmeiotic (rSpd and eSpd) stages [8, 13–16, 32]. This technique is based on measurements of chromatin amount and structure detectable using the fluorescent Hoechst DNA dye.…”

“…S2), suggesting that the isolation of populations enriched for these germ cells can be achieved for other mammalian species. Also, discrimination between different meiotic stages, already resolved for mouse [13–15], would broaden the scope of the application of this technique in the field of male reproductive biology.…”

“…S1), a sequential cell gating strategy was applied: debris was excluded based on forward scatter (FSC) versus side scatter (SSC) plot, then singlets were gated by adjusting threshold for FSC pulse width, and finally red/blue Hoechst fluorescence was used to detect different spermatogenic germ cell populations. For samples obtained by mechanical dissociation or enzymatic dissociation with PI staining (guinea pig and mini pig) two intermediate gating steps were introduced as previously described by Gaysinskaya and Bortvin [14] for the mouse: debris was excluded based on FSC versus SSC plot, then singlets were gated by adjusting threshold for FSC pulse width. Live single cells, negative for PI, were gated based on PI red fluorescence and FSC and plotted in histograms of cell counts in relation to measurements of Hoechst blue fluorescence.…”

“…FACS of Hoechst-stained (Ho-FACS) murine male germ cells can discriminate nine germ cell types [8, 13–16]. Hoechst is a vital dye that binds preferentially to poly(d[AT]) sequences in the minor groove of DNA, with secondary binding taking place at higher ratios.…”

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

“…Given the lack of an in vitro system representative of spermatogenesis progression, and the presence of great cellular heterogeneity in testis, studies of male germ cell biology require robust techniques to isolate enriched populations of specific cell types. Fluorescence-activated cell sorting (FACS) has been widely used for this purpose 1,2,3,4,5 , as it provides high yield and purity, and surpasses other isolation methods in the number of germ cell types that it can identify and select 6,7,8 . The principle of flow cytometry analysis is based on the detection of differential light patterns following laser beam excitation of single cells.…”