Anti cd48 fitc

BM cells were collected from tibias and femurs and stained with anti-c-kit PE-Cy7 (BD Biosciences), anti-Sca-1 APC-Cy7 (BD Biosciences), anti-lineage V450 (BD Biosciences), anti-CD48 FITC (Biolegend), anti-CD127 FITC (BD Biosciences), anti-CD41 Alexa Fluor 488 (Biolegend), anti-CD34 APC (BD Biosciences), anti-CD135 PE (BD Biosciences), anti-CD16/32 PerCP (BD Biosciences) and anti-CD150 Alexa Fluor 647 (Biolegend). BM HSCs (CD150⁺CD48^-Lineage^-Sca-1⁺c-Kit⁺), MPPs (CD150^-CD48^-Lineage^-Sca-1⁺c-Kit⁺), CMPs (CD34⁺CD16/32^lowCD127^-Lineage^-Sca-1^-c-Kit⁺), GMPs (CD34⁺CD16/32^highCD127^-Lineage^-Sca-1^-c-Kit⁺), MEPs (CD34^-CD16/32^-/lowCD127^-Lineage^-Sca-1^-c-Kit⁺), MkPs (CD150⁺CD41⁺Lineage^-Sca-1^-c-Kit⁺) and EPs (CD71⁺Ter119⁺) were stained with the indicated cell surface marker antibodies. For intracellular phosphoprotein analysis, BM cells were flushed into serum-free PBS. BM cells were stained with surface marker antibodies, then washed with PBS and fixed for 15 minutes at 4°C, followed by incubation with BD c ytoperm buffer for 30 minutes at room temperature. Rabbit anti-pS6K (Thr389) (Cell Signaling Technology) or anti-pAkt PE (Cell Signaling Technology) were added at 1:50 dilution for 30 minutes. For pS6K staining, cells were incubated with anti-rabbit IgG PE (Cell Signaling Technology) at 1:500 for 30 minutes. Flow cytometric analysis was performed on a FACS CANTO II (BD Biosciences).

Cells from lung, spleen and lymph nodes were incubated with Fc blocker for 10 min at 4 °C, and then stained with viability dye eFluor 780 (eBioscience), anti-CD45-PerCP-Cy5.5, anti-CD11b-APC, anti-F4/80-PE, anti-Ly6C-PerCP-Cy5.5, anti-Ly6G-PE, anti-CX3CR1-FITC, anti-CD4-APC, and anti-CD8-FITC (BioLegend; 1:300 vol/vol dilution) for 30 min at 4 °C. For BM phenotyping, cells were stained for anti-CD19-APC, anti-Ter119-APC, anti-CD11b-APC, anti-Ly6C/G-APC and anti-CD3-APC as lineage markers along with anti-Ly6A/E-APC-Cy7 (Sca-1), anti-CD117-PE-Cy7 (c-kit), anti-CD48-FITC and anti-CD150-PE-Cy5 (SLAM; BioLegend; 1:300 dilution) for Lin⁻Sca-1⁺c-kit⁺ LSK populations and Lin⁻Sca-1⁺c-kit⁺ CD48⁺CD150⁻ MPPs. Data were collected using FACSCanto flow cytometer (BD Bioscience). The intracellular TNF (anti-TNF-PE; 1:600 dilution) and FoxP3 (anti-FoxP3-PE; 1:300 dilution) staining was performed using Fixation Buffer (BioLegend, 420801) and Foxp3 Staining Buffer Set (Thermo Fisher, 00-5523-00) according to the manufacturer’s instruction. Information for all flow antibodies is provided in the Reporting Summary. Flow data were analyzed using FlowJo software (Tree Star).