TY - JOUR
T1 - Isolation of murine spermatogenic cells using a violet-excited cell-permeable DNA binding dye
AU - Yeh, Yu Han
AU - Hu, Mengwen
AU - Nakagawa, Toshinori
AU - Sakashita, Akihiko
AU - Yoshida, Shosei
AU - Maezawa, So
AU - Namekawa, Satoshi H.
N1 - Funding Information:
Lalor Foundation Postdoctoral Fellowship to A.S.; AMED-CREST (JP17gm1110005h0001) to S.Y.; the Research Project Grant by the Azabu University Research Services Division, Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Private University Research Branding Project (2016–2019), Grant-in-Aid for Research Activity Start-up (19K21196), the Takeda Science Foundation (2019), and the Uehara Memorial Foundation Research Incentive Grant (2018) to S.M.; National Institute of Health R01 GM122776 to S.H.N.
Funding Information:
We thank members of the Namekawa, Yoshida, and Maezawa laboratories for their help; Katie Gerhardt for editing the manuscript; Mary Ann Handel for sharing the H1T antibody, the Cincinnati Children’s Hospital Medical Center (CCHMC) Research Flow Cytometry Core for sharing the FACS equipment supported by NIH S10OD023410; Grant-in-Aid for Scientific Research (KAKENHI; 17K07424) to T.N.;
Publisher Copyright:
© 2021 JoVE Journal of Visualized Experiments.
PY - 2021/1
Y1 - 2021/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85099873202&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85099873202&partnerID=8YFLogxK
U2 - 10.3791/61666
DO - 10.3791/61666
M3 - Article
C2 - 33522502
AN - SCOPUS:85099873202
SN - 1940-087X
VL - 2021
SP - 1
EP - 13
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 167
M1 - e61666
ER -