Research On The Best Method For Apoptosis Rate Detection In Different Stages Of Apoptosis

ObjectiveTo explore better techniques for detection of apoptosis rate at different apoptotic stages, providing reference for choosing the proper methods for comprehensive and accurate detection of apoptosis rate.MethodsA mouse model of dexamethasone-induced thymocyte apoptosis in vivo and vitro was used. The apoptosis rate of various stages of apoptosis cell was detected and analyzed by using both flow cytometry, and DNA agarose gel electrophoresis and some other tequniques at different time points after successful induction. In combination with the model of ultra-violet-induced thymocyte death for comparison, the accuracy of various apoptosis detection methods were further evaluated.Results1、 Flow cytometric analysis of thymocytes in the early stage of apoptosis by staining cells with Annexine V and 7-AAD showed not only a high degree of sensitivity, but also efficiency to distinguish apoptotic from necrotic cells, and thus be suitable for accurate estimation of apoptosis rate. But when the apoptotic cells were in the later stage, especially when the cell membrane was seriously damaged or even broken, it could hardly discriminate the apoptotic cells from the necrotic ones, no it was able to effectively reflect the real rate of apoptotic thymocytes.2、Agarose gel electrophoresis DNA Ladder analysis demonstrated ability for both qualitative and quantitative analysis in each apoptotic stage of the cells, especially for the DNA fragments detection in the late stage after the apoptotic cells have lost their membrane integrity.3、 Both flow cytometry analysis of caspase-3 stained cells and microscopic counting of TUNEL stained slide were applicable for qualitative and quantitative detection of apoptosis as long as cell membrane integrity was intact, although the sensitivity of the caspase-3 analysis was comparatively lower than the Annexine V at the early apoptotic stage, and the TUNEL could produce a higher false positive rate and counting error because of artificial factors.ConclusionsMany apoptosis detection methods are available. For different stages of apoptosis cell, each method has its advantages and disadvantages. To assess the apoptosis rate comprehensively and accurately, the best is to select two or more complementary methods. To those complex sample with uncertain apoptotic status, a combination of Annexine V staining flow cytometry and DNA Ladder quantitative analysis is a considerable good choice.