Pechhold K, Pohl T, Kabelitz D
Department of Immunology, Paul-Ehrlich-Institute, Langen, Germany.
Cytometry. 1994 Jun 1;16(2):152-9. doi: 10.1002/cyto.990160209.
Determination of the number of viable cells or quantification of lymphocyte subsets in heterogeneous cell populations is critically important for cytotoxicity assays, apoptosis assays, or the analysis of differential activation of T-cell subsets by distinct stimuli. In this report, we describe a rapid flow cytometry method termed Standard Cell Dilution Analysis (SCDA) specifically to quantify any subset of phenotypically definable, viable cells in heterogeneous populations using a FACScan flow cytometer. This method combines: (1) specific detection of lymphocyte subsets by phycoerythrin-conjugated monoclonal antibodies, (2) electronic exclusion of dead cells or cell debris by propidium-iodide staining and gating on forward vs. sidescatter, respectively, and (3) admixture of a known amount of fixed, fluorescein isothiocyanate stained cells immediately before analysis as a constant parameter to allow for calculation of cell quantity. We have used SCDA to analyze the in vitro growth characteristics of various human T-lymphocyte subpopulations in response to different activation stimuli.
在异质性细胞群体中确定活细胞数量或定量淋巴细胞亚群，对于细胞毒性测定、凋亡测定或分析不同刺激对T细胞亚群的差异激活而言至关重要。在本报告中，我们描述了一种快速流式细胞术方法，称为标准细胞稀释分析（SCDA），专门用于使用FACScan流式细胞仪对异质性群体中任何表型可定义的活细胞亚群进行定量。该方法结合了：（1）用藻红蛋白偶联的单克隆抗体特异性检测淋巴细胞亚群；（2）通过碘化丙啶染色分别对前向散射和侧向散射进行设门，以电子方式排除死细胞或细胞碎片；（3）在分析前立即混入已知数量的固定的、异硫氰酸荧光素染色的细胞作为恒定参数，以便计算细胞数量。我们已使用SCDA来分析各种人类T淋巴细胞亚群在不同激活刺激下的体外生长特性。
