O'Brien M C, Bolton W E
Immunology Research and Technology, Coulter Corporation, Miami, Florida 33116-9015, USA.
Cytometry. 1995 Mar 1;19(3):243-55. doi: 10.1002/cyto.990190308.
Dead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining. To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells from each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye styryl (LDS)-751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep-alpha-mouse-FITC (SAM-FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specific staining of dead cells strong enough to withstand the permeabilization procedure, high signal-to-noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO-PRO-3, LDS-751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin-SAM-FITC, EMA, and 7-AAD did not display sufficiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every respect other than its specificity only for cells of epithelial origin. Tubulin-SAM-FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe.
死细胞是活细胞流式细胞术分析中一个重要的干扰源，主要原因是非特异性摄取探针、自发荧光增加以及抗原表达和DNA含量改变。基于光散射或摄取碘化丙啶（PI）或荧光素二乙酸酯（FDA）等染料的传统死细胞排除方法，适用于新鲜、相对均匀的样本分析。然而，它们与本实验室开发的包含表面和细胞内联合染色的实体瘤单克隆抗体面板不兼容：在实体瘤等异质样本中光散射不可靠，并且由于结合较弱或可逆，大多数广泛使用的活力探针与用于细胞内染色的通透剂不兼容。为了确定包含在实体瘤面板中的最佳活力标记物，我们比较了收获后在缺氧条件下保存长达15天的培养细胞，用八种活力探针染色，并按照实体瘤面板程序进行处理（每天未处理的细胞用PI染色作为标准）。活力探针包括PI（处理和未处理样本中）；7-氨基放线菌素D（7-AAD）；TO-PRO-3；激光染料苯乙烯基（LDS）-751；叠氮溴乙锭（EMA）；以及用羊抗鼠FITC（SAM-FITC）间接标记的肌动蛋白、细胞角蛋白和微管蛋白。最佳活力探针的选择标准包括广泛的细胞类型特异性：活细胞的非特异性染色低，死细胞的特异性染色强到足以承受通透化程序，在整个时间过程中具有高信噪比，以及与构成肿瘤抗体面板的其他四种荧光探针兼容。TO-PRO-3、LDS-751和PI（处理细胞中）对活细胞和死细胞均无差别染色。肌动蛋白-SAM-FITC、EMA和7-AAD在整个时间过程中未显示出足够高的信噪比。细胞角蛋白-SAM-FITC在各方面都可以接受，只是其仅对上皮来源的细胞具有特异性。单独的微管蛋白-SAM-FITC满足所有标准，并被选作活力探针包含在单克隆抗体面板中。
