Isolation of murine spermatogenic cells using a violet-excited cell-permeable DNA binding dye

Yu Han Yeh, Mengwen Hu, Toshinori Nakagawa, Akihiko Sakashita, Shosei Yoshida, So Maezawa, Satoshi H. Namekawa

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Article numbere61666
Pages (from-to)1-13
Number of pages13
JournalJournal of Visualized Experiments
Issue number167
Publication statusPublished - 2021 Jan
Externally publishedYes

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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