细胞内钙离子指示剂的光致荧光增强

目的 钙离子(Ca2+)指示剂的光学稳定性对于显示细胞胞浆内Ca2+浓度的时间特性非常重要，定量研究Ca2+指示剂的光致荧光增强现象。方法 分别用5种不同功率的光照射5种不同细胞系（MC3T3-E1、RAW264.7、MLO-Y4、MEF3T3、HEK293），研究两种Ca2+指示剂Fluo-4 AM和Oregon green的光致荧光响应。观察光致荧光增强以及随后发生的荧光淬灭，并进一步利用毒胡萝卜素（thapsigargin, TG）刺激引起钙响应峰，分析响应峰的特征参数。结果 高功率光对于Fluo-4 AM或Oregon green都可引起荧光增强现象，但Oregon green染色细胞的响应比例以及光致响应峰的大小和时间跨度都显著小于Fluo-4 AM染色细胞。结论 使用低功率光照射Oregon green染色细胞显示细胞内Ca2+浓度变化具有更好的光学稳定性。
Objective As the photostability of calcium ions (Ca2+) indicators is an important property for indicating the temporal features of cytosolic Ca2+ in cells, this study aims to quantitatively measure the light-induced fluorescence enhancement in cells stained with Ca2+ indicators. Methods Five cell lines, MC3T3-E1, RAW264.7, MLO-Y4, MEF3T3 and HEK293, were exposed to the light with five levels of optical power, respectively, so as to investigate the light induced responses of two commonly-used Ca2+ indicators, Fluo-4 AM and Oregon green. The light-induced fluorescence enhancement, the succeeding photobleaching and the thapsigargin (TG)-induced responsive peak followed by were observed. The characteristic parameters of responsive peaks were further analyzed. Results Light with higher power level would induce the fluorescence enhancement for both Fluo-4 AM or Oregon green, while the responsive percentage as well as the magnitude and time span of light-induced peak of Oregon green-stained cells were significantly lower than those of Fluo-4 AM-stained cells. Conclusions The use of Oregon green with low power level light shows better photostability to indicate the intracellular Ca2+.