Hba pe

Cells were washed twice with PBS and incubated in FACS-buffer (1% bovine serum albumin) with surface antibodies against CD71-VB405 (Miltenyi Biotec; 1:200), CD235-PE (Acris; 1:2500), CD14-APC (Miltenyi Biotec; 1:50), CD16-PE (BD Biosciences; 1:80); CD36-FITC (Pelicluster, Amsterdam, The Netherlands; 1:100); CD34-APC (IQ products, Groningen, the Netherlands; 1;10). Isotype controls were IgG1k-FITC (biolegends), IgG1-PE (Diaclone); IgG1k-APC (eBioscience). For hemoglobin staining on erythrocytes cells were fixed with 0.025% glutaraldehyde (sigma-Aldrich) and 0.5% paraformaldehyde (Signa-Aldrich) in PBS and permeabilized with 0.5% NP40 (Sigma-Aldrich) prior to staining HbA-PE (Santa Cruz; 1:1000) and HbF-APC (Invitrogen; 1:1000).

Erythrocytes were diluted to 1 × 10⁸ cells/ml and incubated with primary antibody (Supplementary Table 3) for 30’ at RT and washed five times with PBS supplemented with 0.5% bovine serum albumin (BSA). Unconjugated antibodies were additionally stained with a secondary antibody goat anti-human APC and incubated for 30’ on ice. Cells were washed once with ice-cold PBS/0.5% BSA. For intracellular staining of globin subunits, cells were fixed using 0.025% glutaraldehyde for 10 min, pelleted (1800 rpm, 5 min) and permeabilized using 0.05% NP40 for 10 min. After pelleting and washing using ice-cold PBS, cells were stained using HbA-PE (Santa Cruz) and HbF-APC (Invitrogen) for 30 min at 4 degrees Celsius. Samples were measured using a Flow Cytometer Canto II (BD Biosciences) and analysed using Flowjo software (Flowjo, LLC, USA).