An improved flow cytometric assay for the determination of cytotoxic T lymphocyte activity
An improved flow cytometric assay for the determination of cytotoxic T lymphocyte activity
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DOI：
10.1016/s0022-1759(01)00507-5
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发表时间：
2002-01-01
影响因子：
2.2
通讯作者：
Mackensen, A
中科院分区：
文献类型：
--
作者：
Fischer, K;Andreesen, R;Mackensen, A
T淋巴细胞、自然杀伤细胞和淋巴因子激活的杀伤细胞的细胞毒活性通常通过放射性测定来测试，该测定检测垂死细胞的质膜崩解后细胞质内容物的释放。与细胞毒性的这种间接评价相反，我们在这里描述了一种改进的荧光测定法，其基于在单细胞水平上对细胞损伤的直接定量和定性流式细胞术分析。靶细胞用PKH-26染色，PKH-26是一种亲脂性染料，其稳定地整合到细胞膜中并允许区分靶细胞和效应细胞。体外孵育3 h后，用AnnexinV-FITC（ann-FITC）和碘化丙啶（PI）共染色，可区分活细胞、早期凋亡细胞和坏死细胞。首先通过对PKH-26阳性靶细胞设门进行数据分析，然后分析ann-FITC和PI阳性亚群。通过减去在不含效应细胞的适当对照中测量的非特异性ann-FITC或PI阳性靶细胞，计算PKH-26门控细胞群中的细胞毒性百分比。靶细胞如原代黑色素瘤细胞或白血病母细胞的膜染色显示PKH-26的高且稳定的负载，而不改变细胞的活力或免疫原性。使用在体外产生的抗原特异性细胞毒性T淋巴细胞（CTL），我们可以证明，这种流式细胞术检测是敏感的，并与标准的Cr-51释放测定相关。结论：改良荧光法是一种简便、重复性好的评价T细胞特异性细胞毒性的方法。(C)2002 Elsevier Science B. V.保留所有权利。
The cytotoxic activity of T lymphocytes, natural killer and lymphokine-activated killer cells is usually tested by radioactive assays, which detect the release of cytoplasmic contents after plasma membrane disintegration of dying cells. In contrast to this indirect evaluation of cytotoxicity, we describe here an improved fluorescence assay that is based on the direct quantitative and qualitative flow cytometric analysis of cell damage at a single cell level. Target cells are stained with PKH-26, a lipophilic dye that stably integrates into the cell membrane and permits distinction between target and effector cells. After 3 h of in vitro incubation, costaining with AnnexinV-FITC (ann-FITC) and propidium iodide (PI) permitted discrimination between vital, early apoptotic and necrotic cells. Data analysis is performed first by gating on PKH-26-positive target cells followed by the analysis of ann-FITC- and PI-positive subpopulations. The percentage of cytotoxicity in the PKH-26-gated cell population is calculated by subtracting non-specific ann-FITC- or PI-positive target cells, measured in appropriate controls without effector cells. Membrane staining of target cells such as primary melanoma cells or leukemic blasts revealed high and stable loading of PKH-26 without altering the viability or the immunogenicity of the cells. Using in vitro-generated antigen-specific cytotoxic T lymphocytes (CTL), we could demonstrate that this flow cytometric assay is sensitive and correlates well with the standard Cr-51 release assay. In conclusion, the improved fluorescence assay is a simple and highly reproducible procedure for evaluating the specific cytotoxicity of T cells. (C) 2002 Elsevier Science B.V. All rights reserved.