Add more 2.5 L of Hoechst or 1 L of PI to each tube (second and third). Prepare a double-stained tube. FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), main (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can become performed in less than 2 h. As reduced processing time is vital to preserve the physiology of cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models. system representative of spermatogenesis progression, and the presence of great cellular heterogeneity in testis, studies of male germ NSC-207895 (XI-006) 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 additional isolation methods in the number of germ cell types that it can determine and select 6,7,8. The basic principle of circulation cytometry analysis is based on the detection of differential light patterns following laser beam excitation of solitary cells. Like a cell passes through the laser it displays/scatters light whatsoever perspectives, proportional to cell size (ahead scatter; FSC) and to intracellular difficulty (part scatter; SSC). Observe Ormerod 9 for detailed information on circulation cytometry. Male germ cells undergo specific modifications in DNA content material, chromatin structure, size and shape throughout different phases of spermatogenesis. Thus, unique cell populations can be recognized and separated by combining light scattering and DNA staining with fluorescent dyes 10,11. Several dyes can be used for this purpose (examined in Geisinger and Rodriguez-Casuriaga 3), such NSC-207895 (XI-006) as Hoechst-33342 (Hoechst) which has been frequently used in circulation cytometry analysis of testicular cells for the past decade 1,2,4,10,12. Upon excitation with UV-light, Hoechst emits blue fluorescence proportional to NSC-207895 (XI-006) the cellular DNA content material whereas far reddish fluorescence displays variability in chromatin structure and compaction 1,13,14. As a result, male germ cells in different phases of differentiation show specific patterns during FACS of Hoechst-stained solitary cell suspensions (Ho-FACS; 1,12). Interestingly, due to a mechanism of dye efflux that is only active during the spermatogonial stage, intensity of Hoechst blue fluorescence is not proportional to chromatin content material in these cells, and they cluster like a part human population during Ho-FACS 15. Additionally, combining Hoechst staining with the non-permeant dye propidium iodide (PI) allows users to discriminate live (PI bad) from deceased (PI positive) cells during FACS 1,2,10,12. This strategy has been previously used in circulation cytometric analyses of testicular germ cells and optimized extensively in the mouse to discriminate up to 9 germ cell types, including cells in 4 different phases of meiosis I PKP4 1,2,4,16. For the purpose of this work, Hoechst staining offers three main advantages. First, Ho-FACS has been successfully applied to the isolation of male germ cells in the mouse model 1,2,12, and additional rodents such as rat and guinea pig 17,18,19. Second, Hoechst is definitely a cell-permeant dye and does not require NSC-207895 (XI-006) membrane permeabilization, so it preserves cell integrity. Finally, no RNase treatment is required since Hoechst binds preferentially to poly(d[AT]) DNA sequences 1,20, which means that RNA is maintained and, in addition to DNA and proteins, can be utilized for further downstream molecular studies of germ cell differentiation. Despite the similarity in DNA ploidy and/or stainability observed in circulation cytometry analyses of mammalian varieties (examined in Geisinger and Rodriguez-Casuriaga 3), there has been a good deal of variability in the protocols explained for male germ cell isolation by circulation cytometry. Different studies have employed specific protocols for cells dissociation, and used unique DNA-binding dyes (only or in combination) and FACS gating strategies in different model organisms, mainly the mouse, rat and guinea pig. Hence, direct assessment of data collected for different varieties can be affected by unaccounted technical artifacts resulting from variability between methodologies. Importantly, the impressive conservation of chromatin dynamics throughout mammalian spermatogenesis (2N-4N-2N-1N) suggests that NSC-207895 (XI-006) a standardized protocol could be transversely applied.