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

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.

Original languageEnglish
Article numbere61666
Pages (from-to)1-13
Number of pages13
JournalJournal of Visualized Experiments
Volume2021
Issue number167
DOIs
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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