BM enriched CD34+ cells were stained with CD34-FITC, CD38-APC/Cy7, CD71-PE, CD33-PE/Cy7, and CD84-APC and sorted for our putative GATA-1 high erythroid progenitor subpopulation. We also stained and sorted cells for the CD123- MEP population (CD34-FITC, CD38-BV421, CD45RA-AF700, CD10-BV650, CD123-PECy7) (Akashi et al., 2000 (link)). Antibody panels in Table S2 . For viability, 7-amino actinomycin D (7-AAD) was used. Our putative population corresponding to GATA-1 high BM progenitors were gated as singlet, viable CD34+, CD38+, CD84hi, CD71hi, CD33− cells. Cells were sorting using a BD FACS Aria II (BD Biosciences) and collected in IMDM 2% FBS for further CFU assays.
CFU assays were performed using the MethoCult™ H4435 Enriched (STEMCELL Technologies). Briefly, progenitor BM sorted cells were seeded (250 or 500 cells/well) into 6 well SmartDish™ (STEMCELL Technologies). After incubation for 14 days, at 37°C in 5% CO2, hematopoietic colony-forming unit were automated counted and analyzed by STEMvision™ Human (STEMCELL Technologies). Differentiation frequency was calculated for each sorted population by number of resulting colonies/numbers of starting cells seeded.
CFU assays were performed using the MethoCult™ H4435 Enriched (STEMCELL Technologies). Briefly, progenitor BM sorted cells were seeded (250 or 500 cells/well) into 6 well SmartDish™ (STEMCELL Technologies). After incubation for 14 days, at 37°C in 5% CO2, hematopoietic colony-forming unit were automated counted and analyzed by STEMvision™ Human (STEMCELL Technologies). Differentiation frequency was calculated for each sorted population by number of resulting colonies/numbers of starting cells seeded.