Establishment And Primary Application Of A Novel High-throughput Quantitative Detection Method Of Apoptosis

Large-scale animal cell culture is used extensively in biotechnology and pharmaceutical industries, during which apoptosis accounts for most of cell deaths. In the past few years, researches have focused on the prevention and control of apoptosis, to which early detection is essential.In our research, DNA-intercalant dyes YO-PRO-l(YP) and PI were used to stain hybridoma cells G7. The staining pattern resulting from the different membrane permeability made it possible to distinguish normal, apoptotic and necrotic cells. Then the apoptotic cells were assessed by fluorescence microscope and flow cytometry. The results obtained above were statistically compared with those from AV/PI flow cytometric assay, which is now commonly accepted as the most specific and sensitive method for apoptosis detection. The comparison indicated that there was significant correlation and no marked difference between the former two and the latter. Therefore, YP/PI staining could be used to substitute for AV/PI flow cytometric assay in the detection of apoptosis. With this understanding, the correlations between the fluorescent intensities of YP/PI and the apoptotic/necrotic cell numbers were confirmed, which made it possible to quantify apoptotic/necrotic cell number by YP/PI intensity. We determined the YP/PI fluorescence intensities of the cells in 96-well plate, under optimized conditions: the final concentrations of YP and PI were 3pM and 4 g/ml respectively; the incubation was at 37 C for 8min; the wavelengths of Ex/Em filters used to measure YP and PI fluorescence were 485/538 and 530/590nm respectively. Combined with the data of cell density by hemocytometer counting or MTT staining, the equations that account for the dependence of apoptotic/necrotic percentages on the fluorescence intensities of YP/PI were derived. This method proved highly sensitive, as it was able to detect as few as 100 apoptotic cells in a sample. Furthermore, apoptosis detection could be easily and quickly carried out in 96 well plates, which made it suitable for high-throughput applications.We also applied the established method to the screening of apoptosis-inducing factor in culture systems and effective anti-apoptosis chemicals. The results showed that the deprivation of glutamine, cystine and glucose and the decrease of pH could induce apoptosis in G7 cell culture. Z-VAD-FMK could effectively inhibit apoptosis in glutamine- or glucose-free cultures, suggesting a potential anti-apoptotic chemical.When exposed to low dose of STS, G7 cells, which were normally round-shaped, exhibited a fibroblast-like elongated cell shape, without significant decrease of viability. We detected the change of cell growth, and cell cycle associated with the shape shift, but the mechanism behind these changes remained unclear.