Flow Cytometry Analysis of Anastasis in Cultured Mammalian Lymphocytes

Apoptosis is a physiological form of programmed cell death characterized by phosphatidylserine externalization, caspase activation, and genomic DNA fragmentation. Recent studies have shown that apoptosis may be reversed even in its late stages, calling the previous notion of a ‘point of no return’ into question. This recovery process has been termed anastasis (‘rising to life’), but its underlying mechanisms are not well understood. Our lab has developed a model of studying anastasis using cultured mouse HT-2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Our previous findings have shown that HT-2 cells accumulate in the G1 stage of the cell cycle following 24 hours of IL2 deprivation and can be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry to investigate which apoptotic events are congruent with anastasis. HT2 cells were deprived of IL2 for 24 hours and then rescued for periods of 0, 24, 48, and 72 hours. CD25 surface expression (high-affinity IL2 receptor) and phosphatidylserine externalization (annexin V staining) were subsequently monitored prior to and following recovery, as markers of T cell activation and early stage apoptosis, respectively. Control HT2 lymphocytes continuously maintained in IL2 comprised a single viable population of CD25^HIGHAnnexin^–cells. Cells deprived of IL2 for 24 hours were observed to fall into two populations: CD25^LOWAnnexin⁺early apoptotic and CD25^LOWAnnexin^-non-apoptotic cells. In both populations, CD25 expression was reduced approximately 10-fold when compared to IL2-maintained controls. During anastasis, three distinct viable cell populations emerged: 1) CD25^LOWAnnexin⁺cells that did not progress into the later stages of apoptosis, 2) CD25^LOWAnnexin^-non-apoptotic cells, and 3) CD25^HIGHAnnexin^-non-apoptotic cells. With advancing recovery time, viable CD25^LOWpopulations decreased as CD25^HIGHcells increased. By 72 hours post-recovery, the majority of cells were actively cycling, annexin negative cells. These findings suggest that IL2-dependent mouse HT-2 lymphocytes may be a promising model to study the mechanisms of anastasis.