Solomon Michael, DeLay Monica, Reynaud Damien
Stem Cell Program, Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, 45229, USA.
Cytek Biosciences, Fremont, California, 94538, USA.
Cytometry A. 2020 Oct;97(10):1057-1065. doi: 10.1002/cyto.a.24041. Epub 2020 May 25.
Phenotypic analysis by flow cytometry is one of the most utilized primary tools to study the hematopoietic system. Here, we present a complex panel designed for spectral flow cytometry that allows for the in-depth analysis of the mouse hematopoietic stem and progenitor compartments. The developed panel encompasses the hematopoietic stem cell (HSC) compartment, an array of multipotent progenitors with early marks of lineage specification and a series of progenitors associated with lymphoid, granulo-macrophagic, megakaryocytic and erythroid lineage commitment. It has a built-in redundancy for key markers known to decipher the fine architecture of the HSC compartment by segregating subsets with different functional potential. As a resource, we used this panel to provide a snapshot view of the evolution of these phenotypically defined hematopoietic compartments during the life of the animals. We show that by using a spectral cytometer, this panel is compatible with the analysis of GFP-expressing gene-reporter mice across the hematopoietic system. We leverage this tool to determine how previously described markers such as CD150, CD34, CD105, CD41, ECPR, and CD49b define specific HSC subsets and confirm that high expression of the transcription factor Gfi1 is a hallmark of the most primitive HSC compartment. Altogether, our results provide a convenient protocol to obtain in one analysis a more extensive view of the hematopoietic architecture in mouse models. Our results could also serve as a base for further development of high-end panels leveraging spectral flow cytometry beyond the 15-fluorochrome panel presented in this report. © 2020 International Society for Advancement of Cytometry.
通过流式细胞术进行表型分析是研究造血系统最常用的主要工具之一。在此，我们展示了一个为光谱流式细胞术设计的复杂组合，可对小鼠造血干细胞和祖细胞区室进行深入分析。所开发的组合涵盖造血干细胞（HSC）区室、一系列具有早期谱系分化标记的多能祖细胞以及与淋巴样、粒巨噬细胞、巨核细胞和红系谱系定向相关的一系列祖细胞。它对已知通过分离具有不同功能潜能的亚群来解读HSC区室精细结构的关键标志物具有内置冗余。作为一种资源，我们使用这个组合来呈现这些表型定义的造血区室在动物生命过程中的演变情况。我们表明，通过使用光谱细胞仪，这个组合与跨造血系统分析表达绿色荧光蛋白（GFP）的基因报告小鼠兼容。我们利用这个工具来确定先前描述的标志物如CD150、CD34、CD105、CD41、ECPR和CD49b如何定义特定的HSC亚群，并证实转录因子Gfi1的高表达是最原始HSC区室的一个标志。总之，我们的结果提供了一个便捷方案，通过一次分析就能更全面地了解小鼠模型中的造血结构。我们的结果也可以作为进一步开发高端组合的基础，利用本报告中未提及的超过15种荧光染料的光谱流式细胞术。© 2020国际细胞计量学促进协会。
