Sorting and characterization of BM-derived murine Flk2⁻CD34⁻LT-HSCs and Flk2⁻CD34⁺ ST-HSCs. (A) Expression analysis of (left) stem cell markers in the Lin⁻ fraction of the BM of C57BL/6 mice. Following mononuclear cell isolation from the BM and lineage depletion, the cells were analyzed for the stem cell markers, Sca-1, c-Kit, CD34, and Flk2; and (right) of Flk2 and CD34 following gating on LSK using flow cytometric analysis. Each data point represents mean ± SD. (Bi) Lin⁻ BM cells were stained for c-Kit and Sca-1 along with CD34 and Flk2. (Bii) Following the selection of live-singlet cells, c-Kit and Sca-1 double-positive cells were investigated for their expression of CD34 and Flk2 to isolate Flk2⁻CD34⁺ HSCs (purple), Flk2⁻CD34⁻ HSCs (green), and progenitors (orange). Gates denote the sorting strategy used for each of the populations (n = 29 independent experiments). (C) The frequency of each sorted population of Flk2⁻CD34⁻ LT-HSCs and Flk2⁻ CD34⁺ ST-HSCs in the whole BM cells (left) and in the Lin⁻ fraction following lineage depletion (right). (D) RNA was isolated from sorted Flk2⁻CD34⁻ LT-HCs and Flk2⁻CD34⁺ ST-HSCs. SYBR green-based real-time qPCR was carried out using primers for CD34 and glyceraldehyde-3-phosphate (GAPDH). Results from qPCR were obtained from n = 3 independent experiments. (Ei) Both HSC populations were stained with CD150 antibody and gated according to fluorescence minus 1 (FMO) to determine CD150 expression using flow cytometric analysis. (Eii) The graph is representative of 1 experiment from n = 3 independent experiments. Results were quantified and analyzed using the unpaired t test (**P = .001) and presented as mean ± SEM.