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目的建立九色流式方法检测人外周血中TCR Vβ淋巴细胞亚群活化及凋亡状态的方案。方法用细胞膜表面抗体CD3、CD4、CD8、CD95、CD69、磷脂结合蛋白Annexin-Ⅴ、TCR Vβ抗体试剂盒和细胞核染料DAPI建立九色流式染色方案。用1例健康成年人外周血标本分别进行抗体滴度实验、最适检测电压选择、单色染色和缺一色染色,确定条件后,对8例健康成年人外周血标本进行检测分析。结果根据细胞分群情况选择了合适的抗体滴度和检测电压,单色染色和缺一色试验确定了适宜补偿,建立了九色流式检测人外周血中TCR Vβ淋巴细胞亚群活化及凋亡状态的方案;采用此方案检测的8例健康成年人标本,TCR Vβ亚群分布与TCR Vβ抗体试剂盒说明书及文献报道分布一致,并能分析各TCR Vβ亚群细胞活化及凋亡状态。结论建立的九色流式方案可检测人外周血中TCR Vβ淋巴细胞亚群活化及凋亡状态,操作简便,结果可靠。
Objective To establish a nine-color flow cytometry assay of peripheral blood TCR Vβ lymphocyte subsets activation and apoptosis of the program. Methods Nine-color flow cytometry was performed using cell surface antibody CD3, CD4, CD8, CD95, CD69, phospholipid-binding protein Annexin-Ⅴ, TCR Vβ antibody kit and nuclear dye DAPI. Antibody titer experiments were performed on peripheral blood samples from 1 healthy adult. The optimal voltage, monochromic and uncolored staining were determined. After the determination of the conditions, the blood samples of 8 healthy adults were detected and analyzed. Results The appropriate antibody titer and detection voltage were selected according to the cell population. Monochromatic staining and uni-color test were used to determine the appropriate compensation. The flow cytometry was used to detect the activation and apoptosis of TCR Vβ lymphocyte subsets in human peripheral blood . The distribution of TCR Vβ subpopulations in 8 healthy adult patients tested by this protocol was consistent with that of the TCR Vβ antibody kit and reported in the literature. The VCR subpopulation activation and apoptosis status of each TCR were also analyzed. Conclusion The established nine-color flow cytometry assay can detect the activation and apoptosis of TCR Vβ lymphocyte subsets in human peripheral blood, with simple and convenient operation and reliable results.