Flow cytometry isolation of murine spermatocytes

To describe in detail an optimized FACS protocol for isolation and enrichment of high-purity individual meiotic prophase I spermatocytes from adult murine testis based on Hoechst 33342 dye staining.
Conclusion:
We present an optimized FACS approach for isolating high-purity individual early and late meiotic prophase I spermatocytes. The optimization was based on significant protocol improvements in tissue dissociation and gating, with incorporation of back-gating and DNA content restriction techniques into the analysis. Specimen Quality Control: Experiments repeated more than fifteen times. Data Acquisition: For each sample, 500,000 to 1 million events were recorder prior to gate setup. Prior to sorting, side streams and drop delay were adjusted using BD AccuDrop beads. The sort setup was done using 70 micron nozzle. Up to several hundred thousand events were. Two-way sorting and “Purity” precision mode in the sort layout were specified. Sorting Flow rate was adjusted to 2000-3500 events/second. Data Analysis: Gates were copied from one experiment to the next, and optimized by manual adjustment for each specimen.