Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)

Abstract This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so‐called side population. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Hoechst is a DNA‐binding dye. It has at least two binding modes that result in different spectral properties, allowing resolution of multiple populations by viewing fluorescence at two wavelengths simultaneously. The ability to discriminate SP cells is based on the differential efflux of the dye by a multi‐drug‐like transporter. Particular attention is given to the critical aspects of Hoechst concentration, cell concentration, staining time, and staining temperature.