Detection of apoptosis of immature CD4⁺8⁺ thymocytes by flow cytometry

Apoptosis, i.e., programmed cell death, may be the mechanism by which both autoreactive and unselected immature CD4⁺8⁺ thymocytes are eliminated in the thymus. In the present paper we describe a simple and rapid flow cytometric method which permits one to study the induction and kinetics of apoptosis of CD4⁺8⁺ thymocytes using in vivo and in vitro suspension cultures. Analysing the level of surface expression of CD4 and CD8 molecules, forward light scatter and side (90°) scatter as well as staining with ethidium bromide, three distinct stages of apoptosis of CD4⁺8⁺ thymocytes were defined. By counting cells passing through different stages of apoptosis one can attempt to quantitate this process. This method should be useful for in vitro studies on the mechanisms of negative and positive selection of CD4⁺8⁺ thymocytes, i.e., induction and inhibition of apoptosis respectively.