Foxp3 permeabilization kit

Mouse blood, spleens and bone marrow (femurs and tibias) were harvested. Bone marrow was processed by grinding with a pestle and mortar. Cells from the bone marrow and spleens were passed through 40-μm filters, washed in FACS buffer (PBS containing BSA 5%) and stained with monoclonal antibodies. Mouse cell stainings were performed to analyze myeloid and progenitor cells (Supplementary table 1). Myeloid cells and monocytes were, respectively, defined as CD19⁻ CD11b⁺ and CD19⁻ Ly6G⁻ CD11b⁺ CD115⁺. HSPC were identified as Lin⁻ c-Kit⁺ Sca-1⁺, GMP as Lin⁻ c-Kit⁺ Sca-1⁻ CD16/32⁺ CD34⁺, MPP as Lin⁻ c-Kit⁺ Sca-1⁻ CD150⁻ CD48⁺ and HSC as Lin- c-Kit⁺ Sca-1⁻ CD150⁺ CD48⁻. Human cell stainings were performed to analyze progenitor cells (Supplementary table 2). Human HSPC population was identified as Lin⁻ CD34⁺ CD38⁻ CD45RA⁻ CD90⁺. For Mtg16 staining, splenic and bone marrow HSC (Lin- c-Kit⁺ Sca-1⁻ CD150⁺ CD48⁻) were permeabilized (eBioscience FoxP3 permeabilization kit, San Diego, CA), incubated with anti CD16/32 Fc block antibody and then stained with mouse anti-Mtg16 (gift from Dr Scott Hiebert) or mouse isotype control.
Data acquisition and analysis were performed using Fortessa Flow Cytometer (Becton Dickinson, Downers Grove, IL) and FlowJo software (Tree Star, Ashland, OR).