The CD86⁻ subset of HSCs efflux Hoechst dye, express high levels of CD150, and are quiescent. (A) CD150⁺ CD48⁻ LSKs were gated and further divided into HSC subsets on the basis of CD86 expression (top left). The bottom panels show moderate-to-high exclusion of Hoechst dye (boxes). The latter population is also known as “side population tip.” HSCs from individual mice in 3 independent experiments were analyzed in this way (n always > 3), and the results are given in the right panel. (B) Absence of CD34 and Flk2 are also defining characteristics of true stem cells, and populations gated in this way include the CD86⁻ subset (contour plots on left). MFIs for CD150 staining are given in the right panel, showing that levels are significantly higher on CD86⁻ HSCs. (C) Ki-67 and DNA staining showed that CD86⁻ HSCs in normal mice are not dividing. Representative plots are shown on the left side, whereas results for 6 individual mice are shown on the right.