Hospital for Sick Children, Program in Developmental and Stem Cell Biology, Toronto, ON, Canada.
Pape Research Institute, Oregon Health & Science University, Portland, OR, USA.
Methods Mol Biol. 2023;2680:157-168. doi: 10.1007/978-1-0716-3275-8_9.
Flow cytometry methods for sorting specific populations of cells based on fluorescence or physical properties have been a widely used technique for decades. Flow cytometry has been particularly vital to the study of planarians, which remain refractory to transgenic transformation, as it has provided a work-around solution for studying stem cell biology and lineage relationships in the context of regeneration. Many flow cytometry applications have been published in planarians, beginning with broad Hoechst-based strategies for isolating cycling stem cells and progressing to more function-based approaches involving vital dyes and surface antibodies. In this protocol, we look to build on the classic DNA-labeling Hoechst staining strategy by adding pyronin Y staining to label RNA. While Hoechst labeling alone allows for the isolation of stem cells in the S/G2/M phases of the cell cycle, heterogeneity within the population of stem cells with 2 C DNA content is not resolved. By considering RNA levels, this protocol can further divide this population of stem cells into two groups: G1 stem cells with relatively high RNA content and a slow-cycling population with low RNA content, which we call RNA stem cells. In addition, we provide instruction for combining this RNA/DNA flow cytometry protocol with EdU labeling experiments and describe an optional step for incorporating immunostaining prior to cell sorting (in this case with the pluripotency marker TSPAN-1). This protocol adds a new staining strategy and examples of combinatorial flow cytometry approaches to the repertoire of flow cytometry techniques for studying planarian stem cells.
基于荧光或物理特性对特定细胞群体进行分选的流式细胞术方法已经成为一种广泛使用的技术，其应用已有数十年之久。流式细胞术对于研究涡虫尤为重要，因为涡虫仍然对转基因转化具有抗性，而流式细胞术为研究干细胞生物学和再生背景下的谱系关系提供了一种解决方法。许多流式细胞术应用已经在涡虫中发表，最初是基于广泛的 Hoechst 策略来分离有丝分裂干细胞，然后发展到更基于功能的方法，包括活细胞染料和表面抗体。在本方案中，我们旨在通过添加吡罗红 Y 染色来标记 RNA，从而扩展经典的 DNA 标记 Hoechst 染色策略。虽然单独的 Hoechst 标记允许分离细胞周期 S/G2/M 期的干细胞，但具有 2C DNA 含量的干细胞群体中的异质性仍未得到解决。通过考虑 RNA 水平，该方案可以将这群干细胞进一步分为两组：具有相对高 RNA 含量的 G1 干细胞和具有低 RNA 含量的慢循环群体，我们将其称为 RNA 干细胞。此外，我们提供了将这种 RNA/DNA 流式细胞术方案与 EdU 标记实验相结合的说明，并描述了在细胞分选前进行免疫染色的可选步骤（在这种情况下，使用多能性标记物 TSPAN-1）。该方案为研究涡虫干细胞的流式细胞术技术组合添加了新的染色策略和组合流式细胞术方法的示例。
