Erythropoietin epo

CD34⁺ cells were cultured using a single-phase ex vivo expansion and differentiation protocol [53 (link)] with minor modifications. Briefly, cells were cultured in IMDM supplemented with 2 U/ml erythropoietin (EPO) (Sigma-Aldrich, Chemie GmbH Germany), 1 μM glucocorticoid dexamethasone (Sigma-Aldrich, Chemie GmbH Germany), 40 ng/ml insulin-like growth factor 1 (IGF-1) (Sigma-Aldrich, South Africa), 100 ng/ml stem cell factor (SCF) (Sigma-Aldrich, Chemie GmbH Germany) and 400 μg/ml holo-human transferrin (Sigma-Aldrich, Germany). The initial seeding density of 0.5−1 × 10⁶ cells/ml were expanded and differentiated to erythroid progenitors for 15 days, with ad hoc demi-population. Post-differentiation, flow cytometry was used to determine expression of CD71 and CD235a (Glycophorin-A). The human erythroleukaemia cell line K562 was cultured in IMDM initially supplemented with 10 % FBS and then 0.5 % in all subsequent experiments.

Erythroblastic cell lines (K562 and HEL 92.1.7) and HeLa cell lines were cultured in RPMI 1640 and D-MEM medium respectively with 10% FBS at 37°C and 5% CO₂, with stimuli being added after replacing the complete medium. Hemin Chloride (Calbiochem) was prepared with sodium hydroxide 0.2 M as a diluent and was used at final concentration of 50 µM. Bafilomycin A1 (Sigma–Aldrich) was prepared in DMSO and diluted in culture medium at a 100 nM. Resveratrol (Sigma–Aldrich) was diluted in Ethanol 70% and used at 100 µM stimulation concentration. Erythropoietin (EPO) (Sigma–Aldrich) was added at a final concentration of 0.2 UN. Lysosomal marker Lysotracker Red (Thermo Fisher Scientific) was incubated for 1 h. For protein synthesis inhibition, 10 µM of cycloheximide (Sigma) was used for 4 h.