Flow cytometric analysis of signaling and apoptosis in hematopoietic stem cells

The analysis of protein phosphorylation in hematopoietic stem cells (HSCs) provides a powerful tool for studying the cell signaling activities that mediate HSC fate decisions, such as self-renewal, differentiation, and apoptosis. The first part of this chapter describes a method of intracellular staining for phosphorylated proteins in conjunction with membrane staining for multiple hematopoietic cell-surface markers, and subsequent flow cytometric analysis of protein phosphorylation levels [indicated by mean fluorescence intensity (MFI) of specific fluorochromed phospho-antibodies] in primitive hematopoietic cells. The second part describes a method for assessing the frequency of apoptosis in HSCs using extracellular staining with recombinant Annexin V and 7-Amino-Actinomycin (7-AAD). Both parts involve an initial magnetic enrichment of hematopoietic stem/progenitor cells from bone marrow. Because of the intracellular detection required for the HSC signaling assay, this assay also includes cell fixation and permeabilization. Gating strategies for assessing MFI and the frequency of Annexin V⁺ apoptotic cells in a complex population are also described along with representative examples.