Pe anti ter119

Mice were injected intraperitoneally with 100 mg/kg phenylhydrazine (PHZ, Sigma). Blood was collected in 3 days after PHZ treatment and stained with FITC-anti-CD71 and PE-anti-Ter119 (Biolegend) for sorting Ter119⁺CD71^high reticulocytes. The sorted cells were cultured in Iscove’s Modified Dulbecco Medium (IMDM, Gibco) supplemented with 30% fetal bovine serum (Hyclone), 1% deionized bovine albumin (Sigma), 100 units/ml penicillin (Gibco), 100 g/ml streptomycin (Sigma) and 0.1 mM α-thioglycerol (Sigma) [15 (link)]. In some experiments, 10 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 0.1 μM wortmannin, 5 αM N-acetyl-l-cysteine (NAC), 5 mU/ml glucose oxidase (GO), or 1 αM o-phenanthroline (Sigma) was also added to the culture. For iron quantification, 1 × 10⁷ cells were collected from the cultures on indicated days and lysed by NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4). The concentration of Fe²⁺ and Fe³⁺ were determined by QuantiChrom Iron Assay Kit (BioAssay Systems) per the manufacturer’ s protocol.