Anti sca1 pe cy7

Each mouse was processed as an individual biological replicate (N = 5, three male, two female). Lineage depletion protocol up to antibody staining was identical to isolation and purification of mouse CMP, GMP and MEPs. After resuspending in FACS buffer (100 μL per sample), Anti-CD34 FITC (clone RAM34; 5.0 μg/mL final concentration) was added. Cells were incubated for 45 min on ice prior to addition of the remaining antibodies: Anti-Lineage Cocktail A700 (3 μL per mouse), Anti-cKIT APC-eFluor780 (clone 2B8; 2 μg/mL final concentration), Anti-Sca1 PE-Cy7 (clone D7; 2 μg/mL final concentration), Anti-CD150 BV421 (clone TC15-12F12.2; 2 μg/mL final concentration), Anti-Flt3 PerCP-eFluor710 (clone A2F10; 2 μg/mL final concentration), Anti-IL7Ra BV711 (clone SB/199, BD Biosciences, San Jose, California; 2 μg/mL final concentration), Anti-CD16/32 PE (clone 93, Biolegend, San Diego, California; 2 μg/mL final concentration), and Anti-ESAM APC (clone 1G8, Biolegend, San Diego, California; 2 μg/mL final concentration). The cells were incubated for an additional 30 min on ice with the complete cocktail prior to washing with FACS buffer (1 mL) to remove excess antibody. Cells were pelleted and resuspended in 250 μL fresh FACS buffer containing DAPI (ThermoFisher Scientific, Waltham, Massachusetts; 1:1000 stock solution) prior to analysis.

Single-cell suspensions from bone marrow or spleen were prepared by passing the tissues through 70 μm cell strainers (BD Pharmingen, San Diego, CA) and red blood cells were lysed in RBC lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA). One million cells from each sample were then labeled with the following, anti-CD41-FITC (BD Pharmingen) or anti-TER119-APC-Cy7 (BD Pharmingen) for 30 minutes on ice. Labeled cells were washed in autoMACS Rinsing Solution (Miltenyi Biotec, Aubum, CA) with the addition of 0.5% BSA, fixed with 1% paraformaldehyde in PBS overnight and then analyzed on a LSRII cytometer (BD Biosciences, San Jose, CA). For analysis of Lineage^-Sca1⁺c-Kit⁺ (LSK) cell and myeloid progenitor cells, ten million bone marrow cells were lineage depleted by using the Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer's protocol with the addition of the anti-IL-7R-biotin antibody (BD Pharmingen). After magnetic separation of the lineage cells through the MACS MS column, the lineage negative cells were incubated with streptavidin-PE-Cy5.5 (eBioscience, San Diego, CA), anti-Sca1-PE-Cy7 (BD Pharmingen), anti-c-Kit-APC (BD Pharmingen), anti-FcγR-PE (BD Pharmingen) and anti-CD34-FITC (BD Pharmingen) on ice for 30 minutes and analyzed on a LSRII cytometer after washing and fixation as described above.