Gating Strategy and Sample Acquisition for Flow Cytometric Identification of Side-population Stem Cells

This protocol describes the application of Hoechst 33342 (bisbenzimide, H 33342; Ho342) staining to identify and purify side-population (SP) stem cells from various human and murine tissue sources (Goodell et al., 1996, 1997; Storms et al., 2000; Jackson et al., 2001). This is a functional assay that uses living cells, allowing investigators to study an active biological process based on the differential efflux of Ho342 by the ABCG2 multidrug transporter (Zhou et al., 2001). UV laser power and alignment, filter settings, sample collection, cell viability, Ho342 concentration, and the staining procedure as a whole are crucial for the optimal resolution of the side population.