Analysis of Apoptosis in Hematopoietic Stem Cells by Flow Cytometry

Analysis of apoptosis can be used to assess aging and survival in the hematopoietic stem cell (HSC) compartment in the context of disease, therapeutic manipulation, or genetic mutation. Two different methods to assess the frequency of apoptosis in the HSC compartment are presented. The first method utilizes an intracellular TUNEL assay that detects DNA strand breaks, a late apoptotic event. The second method relies on an extracellular stain with recombinant AnnexinV that detects flipping of phosphatidylserine groups to the outer membrane leaflet, an early apoptotic event. Both methods involve an initial magnetic enrichment or sorting of hematopoietic stem/progenitor cells from whole bone marrow (BM). Magnetic sorting is followed by polychromatic antibody (Ab) stains that detect AnnexinV or TUNEL staining in the KFTLS or KTLS HSC phenotypes, respectively. Because of the intracellular detection required for the TUNEL assay, that procedure also includes cell fixation and permeabilization. Electronic gating strategies to assess the frequency of AnnexinV⁺ or TUNEL⁺ cells in KFTLS or KTLS HSC phenotypes are also described along with representative examples.