Flow‐cytometric analysis of apoptotic and nonapoptotic T‐cell receptor‐transgenic thymocytes following in vitro presentation of antigen

The use of flow cytometry to detect apoptotic thymocytes is now well established. We have further developed the technique of Hoechst 33342 vital staining to identify discrete stages of murine thymocyte apoptosis (induced by 37 degrees C culture), in conjunction with propidium iodide (PI), cell scatter profile, and surface marker analysis. The first detectable stage was an increase in Hoechst fluorescence without any change in plasma membrane permeability (measured by PI staining). At this early stage thymocytes had already reduced in size, fragmented their DNA, and for the predominant CD4+ CD8+ double positive population, reduced expression of CD4 and CD8. Subsequent to this stage thymocytes continued to reduce in size and decrease expression of CD4 and CD8, though this was accompanied by an increase in membrane permeability. This technique was applied to an in vitro antigen-specific deletion system, where apoptosis of T cell-receptor-transgenic thymocytes was induced upon presentation of self-antigen. Although self-antigen-induced apoptotic thymocytes showed similar characteristics to those undergoing spontaneous apoptosis, there was a significant population of nonapoptotic CD4+ 8+ thymocytes that also had reduced expression of CD4 and CD8. Therefore, we have been able to show that the reduced expression of CD4 and CD8 is not limited to apoptotic thymocytes.