Application of the Ca2+ Indicator Fluo-3 and Fluo-4 in the Process of H2O2 
Induced Apoptosis of A549 Cell

Abstract

Background and objective Lung cancer is a common malignant tumor all over the world, and Ca2+ is a critical regulator for apoptosis of cancer cells. The monitoring of cytoplastic Ca2+ level in real-time will contribute to further investigate the molecular mechanisms of apoptosis mediated by Ca2+ in lung cancer cells. To evaluate the Ca2+ indicator fluo-3 and fluo-4 in the process of H2O2 induced the apoptosis of lung adenocarcinoma A549 cells. The cytoplastic Ca2+ concentration ([Ca2+]i) was determined in real-time, and the correlations between [Ca2+]i and cell apoptosis were investigated. The differences in fluorescence intensity and measured value were compared between the two Ca2+ indicators. Methods Cells were loaded with the Ca2+ indicator fluo-3 or fluo-4 for 1 h, and then stimulated with 50 mM H2O2. Laser scanning confocal microscope was applied to perform real-time monitoring on the variation of [Ca2+]i in selected cells. DAPI staining was used to observe apoptosis in H2O2 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 H2O2 stimulation. The cytoplastic [Ca2+]i was rapidly elevated in H2O2 stimulated A549 cells. The range of [Ca2+]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 H2O2 treated cells, compared with untreated ones (P<0.01). Conclusion In summary, H2O2 promoted Ca2+ release in A549 cells, and induced cell apoptosis. Ca2+ indicator fluo-4 was probably more applicable to measure [Ca2+]i in cells with less content of Ca2+.

Keywords

• Fluo-3
• Fluo-4
• Ca2+
• H2O2
• Apoptosis