An Xiuli, Chen Lixiang
Laboratory of Membrane Biology, New York Blood Center, 310 E 67th St, New York, NY, 10065, USA.
School of Life Science, Zhengzhou University, Science Road 100, Zhengzhou, 450001, P.R. China.
Methods Mol Biol. 2018;1698:193-203. doi: 10.1007/978-1-4939-7428-3_11.
Enucleation is the final step in mammalian erythropoiesis. In this process, the nucleus is extruded by budding off from the erythroblast, forming the reticulocyte. Herein, we describe the flow cytometry-based assays for enucleation assessment. The separation of nucleated erythroblasts, reticulocytes, and extruded nuclei by flow cytometry is based on DNA staining, surface expression of erythrocyte specific markers, or forward scatter (FSC). The enucleation of murine erythroblasts is assessed by the surface expression of murine erythrocyte marker Ter119 and DNA staining. Three discrete populations that represent nucleated erythroblasts, reticulocytes, and extruded nuclei are defined as HoechstTER119, HoechstTER119, and HoechstTER119, respectively. Another nuclei acid staining dye, SYTO16, is used for the assessment of human enucleation in combination with FSC. For human cells, the three populations that represent nucleated erythroblasts, reticulocyte, and extruded nuclei are identified as FSC SYTO16, FSC SYTO16, FSCSYTO16, respectively.
去核是哺乳动物红细胞生成的最后一步。在这个过程中，细胞核从成红细胞上通过出芽的方式被挤出，形成网织红细胞。在此，我们描述基于流式细胞术的去核评估方法。通过流式细胞术分离有核成红细胞、网织红细胞和挤出的细胞核是基于DNA染色、红细胞特异性标志物的表面表达或前向散射（FSC）。小鼠成红细胞的去核通过小鼠红细胞标志物Ter119的表面表达和DNA染色来评估。代表有核成红细胞、网织红细胞和挤出细胞核的三个离散群体分别定义为HoechstTER119、HoechstTER119和HoechstTER119。另一种核酸染色染料SYTO16与FSC结合用于评估人类去核情况。对于人类细胞，代表有核成红细胞、网织红细胞和挤出细胞核的三个群体分别鉴定为FSC SYTO16、FSC SYTO16、FSCSYTO16。
