Apoptosis or programmed cell death is a haracteristic mode of cell death that requires the expression of specific set of genes and has become one of the main issues in field of oncology. Apoptosis is usually detected by its characteristic
morphology
under electron microscopy or evaluated by electrophoretic methods which measure DNA fragmentation in the nuclear extracts. But these techniques are unable to determine, the percent of apoptotic nuclei or recognize the apoptotic cells in a
heterogeneous
cell population. So other methods such as in-situnick translation assay using TdT or flowcytometric analysis have been developed recently and still being investigated.
To further study the method of flowcytometric detection of apoptosis, authors isolated thymocytes from 2~4 weeks old rats, induced apoptosis by treatment with dexamethasone and analysed the cells using fluorescence activated flowcytometry. After
incubation with dexamethasone, a population of cells with reduced DNA content could be observed in DNA frequency histogram and the induction of this population was both time and steroid concentration dependent. This finding was consistent with
endonucleolysis which is the biochemical hallmarker of apoptosis. The position of sub-G0/G1 peak was shifted to lower DNA content value when the cells were maintained in suspension prior to measurement, suggestive of DNA extraction from these
cells.
There were changes in light scattering properties of incubated cells with diminished forward scattering and minimal change of side scatterng. These changes were consistent with chromatin condenstation, the morphologic characteristic of apoptesis.
Cell
viability after dexamethasone treatment was greater than 95%, indicating that the observed changes in dye binding and light scattering property occurred before cell death.
In conclusion, flowcytometric detection of apoptosis is rapid, simple and reproducible and seems to be useful clinically to detect and quantify apoptosis in tumor tissues.
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