Flow cytometric analysis, HPLC, and Wright-Giemsa staining

For HbF and differentiation analysis, 1.5 million cells at day 15 of culture were stained with Live/Dead Fixable Near-IR Dead Cell Stain Kit ({"type":"entrez-nucleotide","attrs":{"text":"L34976","term_id":"522219","term_text":"L34976"}}L34976; ThermoFisher), per the manufacturer’s specifications, then washed in phosphate-buffered saline. Subsequently, the cells were fixed with 0.05% glutaraldehyde (Sigma-Aldrich), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich), and stained with APC-HbF (MHFH05; Invitrogen), PE-CD71 (555537; BD Biosciences), and PECy7-CD235a (306620; Biolegend) at 1:100 dilution. Flow cytometry was performed on a BD FACSCanto system. Cation-exchange HPLC for quantification of HbF and Wright-Giemsa stains for erythroid morphology were performed as previously described.^7,8 Cytospin images were captured at 10× resolution on an Olympus BX60 microscope with Infinity software (Lumenera Corporation).