Griffioen A W, Rijkers G T, Keij J, Zegers B J
Department of Immunology, University Hospital for Children and Youth Het Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands.
J Immunol Methods. 1989 Jun 2;120(1):23-7. doi: 10.1016/0022-1759(89)90284-6.
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 that 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.
膜受体交联后细胞质游离离子钙[Ca2+]i水平升高是T淋巴细胞和B淋巴细胞激活过程中的关键第二信使。荧光钙螯合剂如喹啉-2和吲哚-1的出现，使得准确测量[Ca2+]i成为可能。此类研究中普遍使用的荧光分光光度法的一个主要缺点是记录的是细胞悬液的整体反应。这些数据会受到无反应细胞比例的影响，而无反应细胞的比例会因细胞群体的纯度和所施加刺激的性质不同而有所差异。利用一种基于简单汞弧灯的流式细胞仪——荧光激活细胞分选分析仪（FACS分析仪），已经开发出一种在单细胞水平上测量吲哚-1负载细胞内游离钙反应的准确可靠技术。使用该技术我们发现，刀豆蛋白A激活后T细胞中[Ca2+]i的快速升高（30秒内）仅涉及少数细胞，而所有T细胞在2 - 3分钟内[Ca2+]i水平都会升高。
