Anti lineage cocktail a700

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.

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 mouse), Cells were incubated on ice for 30 min with the following 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-Flt3 PerCP-eFluor710 (clone A2F10; 2 μg/mL final concentration), and Anti-IL7Ra APC (clone A7R34, BD Biosciences, San Jose, California; 2 μg/mL final concentration). Upon incubation completion, cells were diluted with FACS buffer (10 mL) to remove excess antibody. Cell were pelleted (1,300 rpm x 5 min, 4°C) and resuspended in 500 μL/mouse fresh FACS buffer containing SYTOX Blue (1:3000) prior to FACS. Samples were sorted on a BD FACS Aria.