MEASUREMENT OF CYTOPLASMIC CALCIUM IN LYMPHOCYTES USING FLOW-CYTOMETRY - KINETIC-STUDIES AND SINGLE CELL ANALYSIS
MEASUREMENT OF CYTOPLASMIC CALCIUM IN LYMPHOCYTES USING FLOW-CYTOMETRY - KINETIC-STUDIES AND SINGLE CELL ANALYSIS
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DOI：
10.1016/0022-1759(89)90284-6
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发表时间：
1989-06-02
影响因子：
2.2
通讯作者：
ZEGERS, BJM
中科院分区：
文献类型：
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作者：
GRIFFIOEN, AW;RIJKERS, GT;ZEGERS, BJM
膜受体交联后细胞质游离钙 [Ca2+]i 水平升高，是 T 和 B 淋巴细胞激活的关键第二信使。荧光钙螯合剂（例如 quin-2 和 indo-1）的出现使得 [Ca2+]i 的精确测量成为可能。荧光分光光度法是此类研究中常用的方法，其主要缺点之一是记录细胞悬浮液的总体响应。这些数据会因无反应细胞的比例而产生偏差，该比例会根据细胞群的纯度和所施加的刺激的性质而有所不同。使用基于汞弧灯的简单流式细胞仪（FACS 分析仪）开发了一种准确可靠的技术，可在单细胞水平上测量 indo-1 负载细胞中的细胞内游离钙反应。使用这种技术，我们发现，ConA 激活后 T 细胞中 [Ca2+]i 的快速增加（30 秒内）涉及少数细胞，而所有 T 细胞在 2-3 分钟内都显示出 [Ca2+]i 水平增加。
An increased level of cytoplasmic free ionized calcium [Ca2+]i after crosslinking of membrane receptors is a critical second messenger in the activation of T and B lymphocytes. The availability of fluorescent calcium chelators, such as quin-2 and indo-1, makes accurate measurement of [Ca2+]i possible. One of the major drawbacks of spectrofluorometry which is the generally used method in such studies is the overall response of a cell suspension is recorded. Such data will be biased by the proportion of non-responding cells, which will differ according to the purity of cell populations and the nature of the stimulus applied. An accurate and reliable technique to measure intracellular free calcium responses in indo-1-loaded cells at the single cell level has been developed using a simple mercury arc lamp-based flow cytometer, the FACS analyzer. Using this technique we have found that the rapid increase in [Ca2+]i (within 30 s) in T cells following activation by ConA involves a minority of cells, whereas all T cells show increased [Ca2+]i levels within 2-3 min.