应用Ca(2+)荧光探针fluo-3和fluo-4测定H(2)O(2)诱导的A549细胞凋亡过程中的[Ca(2+)](i)变化

BACKGROUND AND OBJECTIVE: Lung cancer is a common malignant tumor all over the world, and Ca(2+) is a critical regulator for apoptosis of cancer cells. The monitoring of cytoplastic Ca(2+) level in real-time will contribute to further investigate the molecular mechanisms of apoptosis mediated by Ca(2+) in lung cancer cells. To evaluate the Ca(2+) indicator fluo-3 and fluo-4 in the process of H(2)O(2) induced the apoptosis of lung adenocarcinoma A549 cells. The cytoplastic Ca(2+) concentration ([Ca(2+)](i)) was determined in real-time, and the correlations between [Ca(2+)](i) and cell apoptosis were investigated. The differences in fluorescence intensity and measured value were compared between the two Ca(2+) indicators. METHODS: Cells were loaded with the Ca(2+) indicator fluo-3 or fluo-4 for 1 h, and then stimulated with 50 mM H(2)O(2). Laser scanning confocal microscope was applied to perform real-time monitoring on the variation of [Ca(2+)](i) in selected cells. DAPI staining was used to observe apoptosis in H(2)O(2) treated cells. RESULTS: Our results showed that the fluorescence intensity of fluo-4 was stronger than that of fluo-3 in the same condition of dye concentration, loading time and image acquisition parameters before or after H(2)O(2) stimulation. The cytoplastic [Ca(2+)](i) was rapidly elevated in H(2)O(2) stimulated A549 cells. The range of [Ca(2+)](i) in selected cells loaded with fluo-3 was 112.2 nM-1, 069.6 nM, and that in selected cells loaded with fluo-4 was 7.6 nM-505.4 nM. Moreover, the apoptotic rate was significantly increased in H(2)O(2) treated cells, compared with untreated ones (P < 0.01). CONCLUSION: In summary, H(2)O(2) promoted Ca(2+) release in A549 cells, and induced cell apoptosis. Ca(2+) indicator fluo-4 was probably more applicable to measure [Ca(2+)](i) in cells with less content of Ca(2+).