Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye.

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.

分离减数分裂的精母细胞对于研究减数分裂和精子发生的分子机制至关重要。尽管已有利用Hoechst 33342染色结合荧光激活细胞分选的成熟细胞分离方案，但它需要配备紫外激光的细胞分选仪。在此我们描述一种使用DyeCycle Violet（DCV）染料的细胞分离方案，这是一种与Hoechst 33342结构相似的低细胞毒性DNA结合染料。DCV可被紫外激光和 violet激光激发，这提高了设备选择的灵活性，包括不配备紫外激光的细胞分选仪。使用该方案，可以分离出减数分裂前期I的三个活细胞亚群，包括细线期/偶线期、粗线期和双线期精母细胞，以及减数分裂后的圆形精子细胞。我们还描述了一种从小鼠睾丸制备单细胞悬液的方案。总体而言，该过程所需时间较短（根据所需细胞数量为4 - 5小时），这有利于许多下游应用。