Mac 1

Single-cell suspensions of whole BM cells were labeled by incubation on ice for 15 min using a cocktail of the following antibodies: PE-conjugated anti-mouse CD3ε (145-2C11; BD Biosciences), CD4 (L3T4; BD Biosciences), CD8α (53-6.7; BD Biosciences), B220 (RA3-6B2; BD Biosciences), Mac-1 (M1/70; BD Biosciences), Gr-1 (RB6-8C5; eBioscience), and Ter119 (TER-119, eBioscience); FITC-conjugated anti-mouse Sca-1 (E13-161.7; BD Biosciences); and APC-conjugated anti-mouse c-Kit (2B8; BD Biosciences). After cells were washed with PBS, they were incubated with anti-PE microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 15 min. Lin⁺ cells (CD4⁺, CD8α⁺, B220⁺, Mac-1⁺, Gr-1⁺, Ter119⁺ cells) were removed by immunomagnetic selection using a MACS system (Miltenyi Biotec). These negatively selected cells were subjected to FACS using a Moflo XDP cell sorter (Beckman-Coulter) to prepare LSK cells.