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 determin e 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. STUDY DESIGN: MGC-803 cells treated with As2O3 were used as the double-stai ning cell model with Hoechst 33342 as a DNA probe and FIuo-3AM as a Ca2+ in dicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainabil ity with Hoechst 33342 and corresponding fluorescence intensity between liv e 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 tes t. RESULTS: The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intrace llular Ca2+ in apoptotic and intact cells were simultaneously detected in t he same sample. CONCLUSION: We provided a useful method to exactly detect changes in intrac ellular Ca2+ in apoptotic cells, especially in single ones, by confocal mic roscopy and to exclude the artifact effect of necrotic and intact cells.