In diabetes mellitus there is an absolute or relative lack of insulin producing beta-cells. This deficiency is thought to be due, at least in part, to enhanced rates of beta-cell apoptosis. Considering the decisive role of beta-cell apoptosis in the pathogenesis of diabetes, it is of great interest that adequate methods are available for the detection of beta-cell apoptosis and necrosis. For the study of beta-cell apoptosis in vitro the methodological alternatives are plentiful. Particularly interesting among these is vital staining of beta-cells with propidium iodide (PI) and Hoechst 33342 (bisbenzimide) and analysis with fluorescence microscopy. This technique allows identification of different stages of both apoptosis and necrosis and is applicable on both mono-layers and intact islets. Since visual inspection of vital stained cells is time consuming and does not easily generate quantitative data, it is desirable to combine the vital staining technique with flow cytometry. Using this technique, islet cells with a moderate increase in PI fluorescence are mainly apoptotic, as assessed by simultaneous Annexin V staining. Moreover, the number of cells with a moderate increase in PI fluorescence corresponds well to the number of cells with increased caspase-3 activity. Thus, vital staining of islet cells with PI followed by flow cytometry gives a valid estimate of islet cell apoptosis. Finally, it is demonstrated that the rate of apoptosis in rat islet cells in vitro is enhanced by the combination of cytokines IL-1 and IFN-γ, but not by high glucose or the sulfonylurea tolbutamide.
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