{"doi":"10.3791/61666","title":"Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye","abstract":"Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.","journal":"Journal of Visualized Experiments","year":2021,"id":197440,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":12,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9561,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":771440,"name":"Mengwen Hu","orcid":"0000-0002-7480-6432","position":1,"is_corresponding":false},{"id":771441,"name":"Toshinori Nakagawa","orcid":"0000-0001-7620-1720","position":2,"is_corresponding":false},{"id":282080,"name":"Akihiko Sakashita","orcid":"0000-0002-3398-8436","position":3,"is_corresponding":false},{"id":771442,"name":"Shosei Yoshida","orcid":"0000-0001-8861-1866","position":4,"is_corresponding":false},{"id":282081,"name":"So Maezawa","orcid":"0000-0002-9060-2042","position":5,"is_corresponding":false},{"id":282088,"name":"Satoshi H. Namekawa","orcid":"0000-0002-1052-943X","position":6,"is_corresponding":false},{"id":772014,"name":"Yu-Han Yeh","orcid":null,"position":0,"is_corresponding":true}],"reference_count":27,"raw_metadata":null,"created_at":"2026-07-18T23:50:23.532351Z","pmid":"33522502","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}