title: 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.
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland.|Department of Biology, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Valeriya
authors.initials: V
authors.lastname: Gaysinskaya
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland.
authors.forename: Alex
authors.initials: A
authors.lastname: Bortvin
mesh_terms: D000375:Aging; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D008297:Male; D049469:Meiotic Prophase I; D051379:Mice; D059010:Single-Cell Analysis; D013090:Spermatocytes / Q000166:cytology*; D013091:Spermatogenesis; D013194:Staining and Labeling; D013737:Testis / Q000166:cytology
keywords: Hoechst 33342; adult mouse testis; cell sorting; flow cytometry; gating; meiotic prophase I
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