Flow cytometry-based cell cycle measurement of mouse hematopoietic stem and progenitor cells

The balance between the quiescent hematopoietic stem cell (HSC) and the highly proliferative hematopoietic progenitor compartments maintains homeostasis in the hematopoietic system. Therefore, the entry of HSCs into the cell cycle and the rate of proliferation of hematopoietic progenitor cells are fundamental aspects in the field. This chapter describes two intracellular staining methods for DNA and RNA in conjunction with membrane staining for multiple hematopoietic cell-surface markers, and subsequent flow cytometric analysis to determine the cell cycle characteristics of primitive hematopoietic cells. First, the DNA stain Hoechst 33342 and the RNA dye Pyronin Y are used in combination with cell-surface markers to identify the proportion of cells in G(0) and G(1) in hematopoietic stem and progenitor cells. The second details the staining of bromodeoxyuridine incorporated into replicating DNA as a measure for the cycling cell fraction within a specific hematopoietic cell subset.