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红细胞的流式细胞术(FCM)分析和荧光激活细胞分选(FACS)

Flow Cytometry (FCM) Analysis and Fluorescence-Activated Cell Sorting (FACS) of Erythroid Cells.

作者信息

An Xiuli, Chen Lixiang

机构信息

Laboratory of Membrane Biology, New York Blood Center, 310 E 67th St, New York, NY, 10065, USA.

School of Life Science, Zhengzhou University, Zhengzhou, China.

出版信息

Methods Mol Biol. 2018;1698:153-174. doi: 10.1007/978-1-4939-7428-3_9.

Abstract

To study the process of erythropoiesis, it is important to be able to isolate erythroid progenitors and erythroblasts at distinct stages of development. During the past decade, considerable progress has been made on the development of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) methods for the analysis and isolation of both murine and human erythroid cells at distinct stages of erythropoiesis, based on changes in the expression of cell surface markers. A method for the identification of murine BFU-E and CFU-E cells was reported by Flygare et al., by negative selection for Ter119, B220, Mac-1, CD3, Gr1, Sca-1, CD16/CD32, CD41, and CD34 cells, followed by separation based on the expression levels of CD71. We developed an alternative method in which Ter119 is used as an erythroid lineage marker, and in conjunction with CD44 and cell size as differentiation markers, it is possible to unambiguously distinguish erythroblasts at each developmental stage during murine terminal erythroid differentiation. We also developed methods for the analysis and isolation of human erythroid cells at all developmental stages. BFU-E and CFU-E are characterized by CD45GPAIL-3RCD34CD36CD71 and CD45GPAIL-3RCD34CD36CD71 phenotypes, respectively; the combination of GPA, band 3 and α4-integrin are used to isolate erythroid cells at all of the terminal stages of human erythropoiesis, including proerythroblasts, early basophilic, late basophilic, polychromatic and orthochromatic erythroblasts.

摘要

为了研究红细胞生成过程,能够在不同发育阶段分离红系祖细胞和幼红细胞非常重要。在过去十年中,基于细胞表面标志物表达的变化,在流式细胞术(FCM)和荧光激活细胞分选(FACS)方法的开发方面取得了显著进展,用于分析和分离处于红细胞生成不同阶段的小鼠和人类红系细胞。Flygare等人报道了一种鉴定小鼠BFU-E和CFU-E细胞的方法,即对Ter119、B220、Mac-1、CD3、Gr1、Sca-1、CD16/CD32、CD41和CD34细胞进行阴性选择,然后根据CD71的表达水平进行分离。我们开发了一种替代方法,其中Ter119用作红系谱系标志物,并结合CD44和细胞大小作为分化标志物,可以明确区分小鼠终末红系分化过程中每个发育阶段的幼红细胞。我们还开发了分析和分离所有发育阶段人类红系细胞的方法。BFU-E和CFU-E分别以CD45GPAIL-3RCD34CD36CD71和CD45GPAIL-3RCD34CD36CD71表型为特征;GPA、带3和α4整合素的组合用于分离人类红细胞生成所有终末阶段的红系细胞,包括原红细胞、早幼嗜碱性红细胞、晚幼嗜碱性红细胞、多染性红细胞和正染性红细胞。

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