Biotin mouse lineage depletion cocktail

Pacific Blue anti-c-Kit (2B8), phycoerythrin (PE)/Cy7 anti-Sca-1 (E13-161.7), and PE or allophycocyanin (APC)/Cy7 anti-Fcγ R III/II (93) were purchased from Biolegend (Tokyo, Japan). Fluorescein isothiocyanate (FITC) anti-CD34 (RAM34) was purchased from eBioscience (San Diego, CA, USA). Biotin Mouse Lineage Depletion Cocktail, biotin anti-CD127 (B12-1), PE anti-CD71 (C2), and FITC anti-CD11b (M1/70) were purchased from BD Biosciences (Tokyo, Japan). Alexa Fluor 647 anti-c-Myb (phosphor S11) (EP769Y) was purchased from Abcam (Tokyo, Japan). Anti-Myb mouse monoclonal antibody (1-1) was purchased from Millipore (Tokyo, Japan). The availability of clone EP769Y toward mouse c-Myb was confirmed by immunoblot assay using mouse thymocyte lysate (Supplementary Fig. S4b), and the equivalent sensitivity of that toward WT and T572A c-Myb was confirmed by immunoblot assay using His-tagged exogenous proteins and the antibodies against His tag or c-Myb, the clone EP769Y or the clone 1-1 (Supplementary Fig. S5).

BM cells (1 × 10⁷ cells/mL) were prepared in PBS containing 3% FCS, incubated on ice for 15 min with 2.5 μg anti-mouse CD16/CD32 (Fc Block, BD Biosciences) per 10⁷ cells, and then incubated on ice for 15 min with Biotin Mouse Lineage Depletion Cocktail (BD Biosciences) consisting of biotinylated monoclonal anti-mouse CD3e, CD11b, CD45R/B220, Ly-6G, Ly-6C, and TER-119. Subsequently, the mixture was washed with 10 volumes of PBS containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, and centrifuged. The resulting cell pellets were mixed with Streptavidin Particles Plus-DM (BD Biosciences), incubated at 6–12 °C for 30 min, and lineage-positive cells were removed using BD IMagnet (BD Biosciences) according to the manufacturer’s protocol. The depleted fractions were then used as lineage-negative BM cells, which have about 10-fold higher colony-forming activity than crude BM cells.