Perm wash buffer kit

Human or mouse cells were harvested and Fc receptors of cells were blocked with anti-CD16/32 (BioLegend) or human IgG (Sigma-Aldrich) for 10 min followed by staining of cells for 15 min at room temperature. For intracellular staining, cells were fixed in 2% formaldehyde and then permeabilized using the Perm/Wash buffer kit (BD Biosciences) followed by antibody incubation. Monoclonal antibodies used for flow cytometry include the following: anti–c-Kit, anti-Sca1, anti-CD48, anti-CD150, anti-Flt3 ligand, anti-CD3, anti-TER119, anti–Gr-1, and anti-B220 (BioLegend) for mouse studies and anti-CD68 (BioLegend), anti-CD163 (Biolegend), and anti-206 (eBioscience) for human studies. Rabbit antitrimethylated H3K4 (Abcam) and antitrimethylated H3K27 (Millipore) were used to detect histone marks, followed by secondary stain with FITC-AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch). All populations were routinely back gated to verify purity and gating. Samples were analyzed on an LSR II (BD Biosciences). One million viable cells were analyzed. Data were analyzed using FlowJo software version 9.0 (Tree Star, Inc.) and compiled using Prism (GraphPad Software).