Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry.
Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry.
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DOI：
10.4049/jimmunol.137.3.952
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发表时间：
1986-08
影响因子：
4.4
通讯作者：
P. S. Rabinovitch;Carl H. June;Angelika Grossmann;Jeffrey A. Ledbetter
中科院分区：
文献类型：
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作者：
P. S. Rabinovitch;Carl H. June;Angelika Grossmann;Jeffrey A. Ledbetter
细胞内离子钙浓度（[Ca 2 +]i）的测量在阐明[Ca 2 +]i作为细胞对激活刺激的反应的触发器的中心作用中是必不可少的。这些研究采用了染料quin 2，其还不容易适用于单个小细胞的分析。我们在这里表明，大量的单细胞的钙反应，可以使用流式细胞术和最近描述的染料，indo-1进行分析。这样的分析表明，第一次的异质性的[Ca 2 +]i响应有丝分裂刺激内的外周血淋巴细胞（PBL）的群体。通过同时定量的一个或两个颜色的表面免疫荧光标记，这种异质性的[Ca 2 +]i反应在PBL中的一些被证明是相关的细胞免疫表型。几乎所有的T细胞都对抗CD 3抗体有反应;然而，CD 4+细胞的反应大于CD 8+细胞，并且在CD 4+细胞群内，由常见白细胞标志物p220的表达定义的亚群之间对CD 3抗体刺激的反应率不同。相比之下，即使在非常高的剂量下，也不是所有的T细胞都对植物血凝素（PHA）有反应。与抗-CD 3一样，在用PHA刺激后，CD 4+细胞显示出比CD 8+细胞更大比例的应答细胞。在单独的实验中，indo-1被发现不损害PBL的生殖活力，从而提供了通过基于对刺激的[Ca 2 +]i响应进行分选来分离细胞后分析功能活性的潜力。混合实验表明，可以很容易地检测到一个亚群的反应，占总细胞的0.3%。因此，流式细胞仪检测indo-1是第一个允许定量分析细胞的小亚群的反应差异和细胞间[Ca 2 +]i的变化的技术。
Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.