Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques.

Abstract

The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10-nonyl-acridine-orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX-treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct 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 scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability.