Application of the Ca2+Indicator Fluo-3 and Fluo-4 in the Process of H2O2 Induced Apoptosis of A549 Cell
10.3779/j.issn.1009-3419.2014.03.03
- VernacularTitle:应用Ca2+荧光探针fluo-3和fluo-4测定H2O2诱导的A549细胞凋亡过程中的[Ca2+]i变化
- Author:
ZHANG SIYANG
1
;
LI CHUNYAN
;
GAO JIAN
;
QIU XUESHAN
;
CUI ZESHI
Author Information
1. 中国医科大学实验技术中心
- Keywords:
Fluo-3;
Fluo-4;
Ca2+;
H2O2;
Apoptosis
- From:
Chinese Journal of Lung Cancer
2014;(3):197-202
- CountryChina
- Language:Chinese
-
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. hTe 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 lfuo-3 and lfuo-4 in the process of H2O2 induced the apoptosis of lung adenocarcinoma A549 cells. hTe cytoplastic Ca2+concentration ([Ca2+]i) was determined in real-time, and the correlations between [Ca2+]i and cell apoptosis were investigated. hTe differences in lfuorescence intensity and measured value were compared between the two Ca2+indicators. Methods Cells were loaded with the Ca2+indicator lfuo-3 or lfuo-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 apop-tosis in H2O2 treated cells. Results Our results showed that the lfuorescence intensity of lfuo-4 was stronger than that of lfuo-3 in the same condition of dye concentration, loading time and image acquisition parameters before or atfer H2O2 stimulation. hTe cytoplastic [Ca2+]i was rapidly elevated in H2O2 stimulated A549 cells. hTe range of [Ca2+]i in selected cells loaded with lfuo-3 was 112.2 nM-1,069.6 nM, and that in selected cells loaded with lfuo-4 was 7.6 nM-505.4 nM. Moreover, the apoptotic rate was signiifcantly 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 lfuo-4 was probably more applicable to measure [Ca2+]i in cells with less content of Ca2+.
