Esain Virginie, Cortes Mauricio, North Trista E
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115, USA.
Harvard Stem Cell Institute, Cambridge, MA, 02138, USA.
Methods Mol Biol. 2016;1451:191-206. doi: 10.1007/978-1-4939-3771-4_13.
Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.
在过去20年里，斑马鱼已被证明是一种用于剖析造血过程中涉及的信号通路的重要模型，包括造血干细胞和祖细胞（HSPC）的形成及稳态维持。尽管人们付出了巨大努力来开发在体外和体内表征HSPC所需的工具，但斑马鱼研究领域仍缺乏跨实验室定量HSPC的标准化方法。在此，我们描述了三种在我们实验室及其他实验室常规使用的方法，用于可靠地计数斑马鱼胚胎中的HSPC：对转基因报告系进行大规模活体成像、荧光激活细胞分选（FACS）以及体外细胞培养。虽然活体成像和FACS分析能够对总HSPC或位点特异性HSPC进行计数，但细胞培养试验提供了独特的机会来测试分离出的HSPC的功能潜力，这与在哺乳动物中使用的方法类似。
