Analysis of thymic lymphocyte apoptosis using in vitro techniques

Thymocyte apoptosis was analyzed in 4-week-old chicks that were treated with dexamethasone. Glucocorticoid treatment of birds resulted in the internucleosomal cleavage of thymocyte DNA as detected by agarose gel electrophoresis and the generation of a population of thymocyte nuclei with a hypodiploid content of cellular DNA when analyzed by flow cytometry. Visualization of apoptotic thymocytes at the ultrastructural level or via confocal microscopy revealed the typical morphological characteristics of cells undergoing apoptosis. Similar techniques were employed to further analyze apoptosis using an in vitro approach where extracts of thymocyte nuclear proteins from glucocorticoid-treated birds were incubated with chicken red blood cell (cRBC) nuclei. The thymocyte nuclear protein extract contained an endonuclease activity that degraded chromatin at internucleosomal sites and generated an amplified population of hypodiploid cRBC nuclei, similar to that detected in vivo in glucocorticoid-treated thymocytes. These data demonstrate a clear correlation between the detection of apoptotic endonuclease activity generated in vivo, where endogenous thymocyte DNA was degraded, with an in vitro assay, where extracted thymocyte nuclear proteins demonstrated a similar type of nuclease activity when cRBC nuclei were employed as an exogenous chromatin substrate.