title: [Application of the Ca2+ indicator fluo-3 and fluo-4 in the process of H2O2 induced apoptosis of A549 cell].
abstract: 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 Ca2+ in lung cancer cells. To evaluate the Ca(2+) indicator fluo-3 and fluo-4 in the process of H2O2 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 H2O2. 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 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 [Ca(2+)]i was rapidly elevated in H2O2 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 H2O2 treated cells, compared with untreated ones (P<0.01).
  
  
  CONCLUSIONS
  In summary, H2O2 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+).
authors.affiliation: Center of Laboratory Technology and Experimental Medicine, China Medical University,  Shenyang 110001, China.
authors.forename: Siyang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Center of Laboratory Technology and Experimental Medicine, China Medical University,  Shenyang 110001, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Li
authors.affiliation: Center of Laboratory Technology and Experimental Medicine, China Medical University,  Shenyang 110001, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Pathology, China Medical University,  Shenyang 110001, China.
authors.forename: Xueshan
authors.initials: X
authors.lastname: Qiu
authors.affiliation: Center of Laboratory Technology and Experimental Medicine, China Medical University,  Shenyang 110001, China.
authors.forename: Zeshi
authors.initials: Z
authors.lastname: Cui
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D017209:Apoptosis; D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008175:Lung Neoplasms / Q000378:metabolism* / Q000503:physiopathology; D018613:Microscopy, Confocal / Q000379:methods; D014966:Xanthenes / Q000737:chemistry / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D006861:Hydrogen Peroxide; D002118:Calcium