New application of fluorescence correlation spectroscopy for early detection of apoptosis in cells




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In the present study, early stage apoptosis is explored with high temporal resolution. In addition to monitoring early apoptosis induction in single cells by ultrasensitive confocal fluorescence microscopy (UCFM), the mitochondrial proteins release kinetics was explored. The current study shows development and optimization of a novel, rapid apoptosis assay to explore the earliest changes in cells by the intrinsic apoptosis pathway. We show that early apoptotic changes in the mitochondria begin nearly simultaneously with the addition of an apoptosis-inducing drug, such as staurosporine. With a temporal resolution of five minutes, this non-invasive analytical technique can elucidate the earliest apoptotic events in living cells. Moreover, our results show that the mitochondrial inter-membrane proteins are not involved in the extrinsic pathway of Ramos cells mediated by an anti-CD95 antibody. Additional techniques such as light microscopy, flow cytometry and microfluidic technology were employed to explore ultrasensitive and high throughput detection of apoptosis in cells using confocal fluorescence microscopy. The results of this study help to understand the earliest mechanisms of apoptosis induction in cells and to develop high throughput cell screening using microfluidic technology, enabling new methods of rapid drug testing and dose-response analyses.



Fluorescence correlation spectroscopy, Mitochondria, High temporal resolution, High throughput screening, Apoptosis, Mitotracker deep red, Single cell screening, Microfluids