A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342.
OBJECTIVE
To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy.


METHODS
MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test.


RESULTS
The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample.


CONCLUSIONS
We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells.
Zhang
T
T
Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Cao
E H
EH
Li
J F
JF
D000814:Aniline Compounds / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology; D001562:Benzimidazoles / Q000032:analysis; D002118:Calcium / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods*; D010087:Oxides / Q000494:pharmacology; D015203:Reproducibility of Results; D013274:Stomach Neoplasms / Q000378:metabolism / Q000473:pathology*; D014407:Tumor Cells, Cultured; D014966:Xanthenes / Q000032:analysis
D000814:Aniline Compounds; D001152:Arsenicals; D001562:Benzimidazoles; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D010087:Oxides; D014966:Xanthenes; C059715:Fluo-3; C017807:bisbenzimide ethoxide trihydrochloride; D000077237:Arsenic Trioxide; D002118:Calcium