Hematopoietic stem cell characterization by Hoechst 33342 and rhodamine 123 staining

A dual-dye efflux strategy utilizing the supravital dyes Hoechst 33342 (Ho) and rhodamine 123 (Rh123) is described and illustrated for the detection and analysis of hematopoietic stem cells in murine bone marrow. Mononuclear cells from bone marrow cell suspensions were incubated in a cocktail of Rh123 plus Ho, and both dyes were effluxed by two 15-min incubations in dye-free buffer prior to sorting. Compared to our original prototype method in which Rh123, but not Ho, was effluxed, this dual-dye efflux protocol more rapidly and efficiently resolves the most primitive Hodull/Rhdull hematopoietic stem cells. Moreover, under conditions of optimal dual-dye uptake and efflux, Hodull/Rhdull cells map to the subfraction of side population (SP) cells with the highest efflux of Ho, which were previously demonstrated to possess the highest hematopoietic stem cell activity.