The effect of cytokines on programd cell death (apoptosis) in ovary.

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Apoptosis is a physiological mode of cell death, observed during procceses such as tissue organization and turnover, or follicle atresia. A fundamental biochemical mechanism of apoptosis is a change in chromatin structure, often associated with fragmentation of DNA into oligonucleosomal fragment of about n x 185 base pairs due to the activation of an endogenous endonuclease. These apoptotic DNA fragments can be visualized as a distinct ladder of DNA band following gel electrophoresis and ethidium bromide staining. But large amount of DNA required for ethidium bromide staining method. So another workers reported autoradiographic method, the DNA is radiolabeled with 32P-ddATP on 3'-ends using terminal deoxy transferase enzyme (TdT). But reduced radioisotope method use offers safety and environmental benefits, simplifies regulatory compliance, and reduce costs for storage and disposal. We tried to establish microscale non-radioactive method for the qualitative and quantitative analysis of … More apoptotic DNA fragmentation. This procedure utilized TdT to uniformly add digoxigenin (DIG) -ddUTP to the 3'-end of DNA fragments. Following gel electrophoresis, labeled DNA was transferred to nylon membrane according to the southern blotting. The membrane was then incubated with the anti-DIG alkaline phosphatase conjugate. After washing the membrane was incubate with CDP-Star (chemiluminescence substrate) for 5 min, sealed in development folder. After 3 or 5 hours incubation, the X ray film was placed on the development folder and exposed 30 sec or 2 min. This method required as little as 200ng of cellular DNA and increase sensitivity of apoptotic DNA detection by at least 20 fold over the widely used ethidium bromide staining method. The total amount of DIG label incorporated into low molecular weight DNA fraction can be quantified and used to estimate the degree of apoptotic DNA fragment by analyzing the X ray film with image analysis software. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells. Less