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 e stablished. We have further developed the technique of Hoechst 33342 v ital staining to identify discrete stages of murine thymocyte apoptosi s (induced by 37 degrees C culture), in conjunction with propidium iod ide (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 t his early stage thymocytes had already reduced in size, fragmented the ir DNA, and for the predominant CD4(+)CD8(+) double positive populatio n, reduced expression of CD4 and CD8. Subsequent to this stage thymocy tes 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 s ystem, where apoptosis of T cell-receptor-transgenic thymocytes was in duced upon presentation of self-antigen. Although self-antigen-induced apoptotic thymocytes showed similar characteristics to those undergoi ng spontaneous apoptosis, there was a significant population of nonapo ptotic CD4(+)8(+) thymocytes that also had reduced expression of CD4 a nd CD8. Therefore, we have been able to show that the reduced expressi on of CD4 and CD8 is not Limited to apoptotic thymocytes. (C) 1994 Wil ey-Liss, Inc.