应用FACS测定细胞激活时胞内钙离子浓度的变化

FACS analysis of changes in intracellular Ca^(++) during cell activation

细胞内钙离子浓度([Ca^(++)]i)的升高,是淋巴细胞激活和增殖过程中早期呈现的一个重要现象。本文报告应用流式细胞仪(FACS)测定[Ca^(++)]i变化的一种方法。Fluo-3是一种荧光活性物质。它在细胞内与游离Ca^(++)结合后,其荧光强度能增高达40倍。因而,可通过FACS分析细胞的荧光强度,来测定[Ca^(++)]i的变化。然后,通过统计[Ca^(++)]i升高的相对细胞数,了解细胞的激活状况。实验摸索了Fluo-3染色的适宜条件。ConA刺激下,Fluo-3染色淋巴细胞呈现典型的[Ca^(++)]i升高的应答。同时应用罗达明标记的抗Thy1单抗区别T细胞和非T细胞及在ConA刺激下[Ca^(++)]i变化的差异。

Elevation of intracellular calcium concentration ([Ca^(++)]i) is a critical early event in the pro-cesses of cell ativation and proliferation. A method to measure the changes of [Ca^(++)]i with FACSis reported. Fluo-3 is a fluorescence dye, whose fluorescence intensity increases 40 folds afterbinding with free Ca^(++). Changes of [Ca^(++)]i, therefor, can be detected by the analysis of fluores-cence intensity of a single cell. The status of cell activation can be demonstrated through elevated [Ca^(++)]i in relative responding cells. The appropriate conditions chosen for doing this assay are al-so described. Typical [Ca^(++)]i mobilization was observed when the Fluo-3-Sfained Cells werestimulated with ConA. Different activation patterns from T lymphocytes and non-T cells wereshowen using Rhodamin-labelled anti-Thyl. monoclonal antibodies.