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目的建立多色流式方法,检测艾滋病病毒(HIV)感染者/艾滋病(AIDS)病人(简称HIV/AIDS病人)中的辅助性T细胞亚群。方法用3种细胞表面抗体CD3、CD4、CD8,6种细胞内因子抗体γ干扰素(IFN-γ)、粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素17(IL-17)、肿瘤坏死因子α(TNF-α)、IL-2、L-10,建立多色流式染色方案。根据多色流式荧光搭配原则,制定最佳配色方案。用1例HIV/AIDS病人外周血标本分别进行最适检测电压调节、荧光补偿设定、减一阴性对照(FMO对照)。确定条件后,对3例HIV/AIDS病人外周血标本进行检测分析。结果根据阴性细胞确定检测电压,用补偿微球(CompBeads)单色染色与细胞单色染色确定适宜补偿,减一阴性对照确定阴性边界,建立了九色流式方法检测HIV/AIDS病人外周血中辅助性T淋巴细胞亚群的方案,在单细胞水平同时检测多个因子。结论建立的多色流式方案可检测HIV/AIDS病人外周血中的辅助性T细胞亚群,能在单细胞水平同时检测多个因子,所需样品少,操作简便、相对快速并能准确定量。
Objective To establish a multicolor flow cytometry method to detect helper T cell subsets in HIV / AIDS patients (HIV / AIDS patients). Methods Three cell surface antibodies, CD3, CD4, CD8 and 6 kinds of intracellular factor IFN-γ, GM-CSF, IL-17 ), Tumor necrosis factor alpha (TNF-alpha), IL-2 and L-10. According to the principle of multi-color flow fluorescence matching, make the best color scheme. One case of HIV / AIDS patients with peripheral blood samples were the best detection voltage adjustment, fluorescence compensation settings, minus one negative control (FMO control). After confirming the condition, the peripheral blood samples from 3 HIV / AIDS patients were detected and analyzed. Results The detection voltage was determined according to the negative cells. CompBads monochromatic staining and monochromatic cell staining were used to determine the appropriate compensation. Negative control was used to determine the negative boundary. Nine-color flow cytometry was used to detect the peripheral blood of HIV / AIDS patients Assisted T-lymphocyte subpopulation programs that simultaneously detect multiple factors at the single cell level. Conclusions The multi-color flow cytometry protocol can detect helper T cell subsets in the peripheral blood of HIV / AIDS patients and can simultaneously detect multiple factors at the single cell level. The sample required is few and simple to operate, relatively fast and accurately quantified .