Schmid I, Hausner M A, Cole S W, Uittenbogaart C H, Giorgi J V, Jamieson B D
Department of Hematology/Oncology, UCLA School of Medicine, 12-236 Factor Building, Los Angeles, CA 90095, USA.
J Immunol Methods. 2001 Jan 1;247(1-2):175-86. doi: 10.1016/s0022-1759(00)00323-9.
Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 1x PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.
通过流式细胞术对单细胞的DNA含量和免疫荧光进行联合分析，可提供混合细胞制剂中细胞亚群增殖反应的信息。然而，大量死亡（无活力）细胞的存在会损害流式细胞术数据分析的准确性，主要原因是死亡细胞可非特异性结合抗体，并在DNA染色图谱上显示出改变。我们开发了一种快速方法，用于在同时进行双色免疫荧光和DNA含量染色的细胞制剂中鉴定死亡细胞。细胞用7-氨基放线菌素D（7-AAD）染色以区分死亡细胞，并用异硫氰酸荧光素（FITC）和藻红蛋白（PE）标记的单克隆抗体（mAb）进行细胞表面免疫荧光染色。细胞通透后，通过添加其非荧光类似物放线菌素D（AD）来阻止7-AAD从染色的死亡细胞扩散到未染色的活细胞中。DNA用红色激发的碘化丙啶（TP3）染色，其发射光谱可与FITC、PE和7-AAD的发射光谱有效分离。TP3染色在含有皂苷（PCBS）的低pH磷酸盐 - 柠檬酸盐缓冲液中于核糖核酸酶A（RNAse）存在下进行。FITC荧光对酸性pH敏感；因此，DNA染色后用含皂苷的pH 7.2的1x PBS替换PCBS，以允许在流式细胞仪上准确检测FITC免疫荧光。我们将此方法应用于低活力的人外周血单个核细胞（PBMC）培养物中淋巴细胞亚群的差异增殖分析。
