{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T01:45:10Z","timestamp":1791164710061,"version":"4.1.0"},"reference-count":33,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2014,3,24]],"date-time":"2014-03-24T00:00:00Z","timestamp":1395619200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2014,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis, and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment, and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells' similar characteristics. In this report, we describe an optimized Hoechst\u201033342 (Hoechst)\u2010based flow cytometry approach for isolating individual MPI populations from adult mouse testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back\u2010gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non\u2010meiotic cells. Finally, we confirm significant enrichment of high\u2010purity Preleptotene (PreL), L, Z, P, and D MPI spermatocytes using stage\u2010specific marker distribution. The technique will facilitate understanding of the molecular events underlying MPI. \u00a9 2014 International Society for Advancement of Cytometry<\/jats:p>","DOI":"10.1002\/cyto.a.22463","type":"journal-article","created":{"date-parts":[[2014,3,24]],"date-time":"2014-03-24T23:31:20Z","timestamp":1395703880000},"page":"556-565","source":"Crossref","is-referenced-by-count":130,"title":["Optimized flow cytometry isolation of murine spermatocytes"],"prefix":"10.1002","volume":"85","author":[{"given":"Valeriya","family":"Gaysinskaya","sequence":"first","affiliation":[{"name":"Department of Embryology Carnegie Institution for Science Baltimore Maryland 21218"},{"name":"Department of Biology Johns Hopkins University Baltimore Maryland 21218"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ina Y.","family":"Soh","sequence":"additional","affiliation":[{"name":"Department of Embryology Carnegie Institution for Science Baltimore Maryland 21218"},{"name":"Department of Biology Johns Hopkins University Baltimore Maryland 21218"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Godfried W.","family":"van der Heijden","sequence":"additional","affiliation":[{"name":"Department of Embryology Carnegie Institution for Science Baltimore Maryland 21218"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex","family":"Bortvin","sequence":"additional","affiliation":[{"name":"Department of Embryology Carnegie Institution for Science Baltimore Maryland 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