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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。
The optical stability of the Ca (2+) indicator of interest is of crucial importance for revealing the time-dependent Ca2 + concentration in the cytoplasm of cells and to quantitatively study the photoluminescence enhancement of Ca2 + indicator. Methods Five different cell lines (MC3T3-E1, RAW264.7, MLO-Y4, MEF3T3 and HEK293) were irradiated with five different light intensities to study the fluorescence of two Ca2 + indicators Fluo-4 AM and Oregon green response. The enhancement of photofluorescence and subsequent fluorescence quenching were observed, and further the peak of calcium response was stimulated by thapsigargin (TG), and the characteristic parameters of response peak were analyzed. Results Fluorescence enhancement was observed in both Fluo-4 AM and Oregon green cells. However, the response ratio of Oregon green-stained cells and the peak size and time span of photoreactive peaks were significantly lower than those of Fluo-4 AM stained cells. Conclusion Oregon green-stained cells irradiated with low-power light show better optical stability with changes of intracellular Ca2 + concentration.