FLOW CYTOMETRIC ANALYSIS OF THE EARLY PHASES OF APOPTOSIS BY CELLULARAND NUCLEAR TECHNIQUES

The early events occurring during apoptosis at the plasma membrane, ch romatin, and mitochondrial levels were investigated in freshly isolate d irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In int act, urfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset sugg estive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphoc yte model. EB could not be substituted for by propidium iodide, indica ting that the nature of DNA probe used is of major importance for dete cting initial apoptotic changes. Because mitochondria are thought to r epresent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and non sensitive (10-nonyl-acridine-orange) probes concomitantly with EB upta ke. Cells starting apoptosis had an enhanced incorporation of both mit ochondria dyes, which in combination with EB identified several cell s ubsets. This suggests that complex alterations in mitochondrial struct ure and functioning occur in the early stages of apoptosis. To investi gate phenomena occurring at the chromatin level In similar phases of a poptosis, irradiated human lymphocytes and DEX-treated murine thymocyt es were disrupted and DNA stainability assessed In nuclear suspensions . A transient increase hn DNA stainability, i.e., the appearance of di stinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations In light s cattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability. (C) 1996 Wil ey-Liss, Inc.