Pe rat anti mouse cd71

To isolate erythroblasts at different stages of maturation by cell sorting, >15 × 10⁶ bone marrow cells from six 7 week-old Sv129/C57BL/6 Fam132b^+/+ littermate mouse femurs (three controls and three 15 hours after phlebotomy) were filtered through a 30 μm filter and resuspended in 1 mL PBS/5% FCS. Cells were blocked with rat anti-mouse CD16/CD32 (0.25 μL/10⁶ cells, BD Biosciences no. 553142) for 15 minutes and subsequently stained with FITC rat anti-mouse TER-119 (0.5 μL/10⁶ cells, BD Biosciences no. 557915), and PE rat anti-mouse CD71 (0.5 μL/10⁶ cells, BD Biosciences no. 553267) and incubated on ice for 30 minutes in the dark. Cells were washed twice with 1 mL PBS and resuspended in 2 mL PBS/5%FCS. Sorting was performed on a FACSAriaII High-Speed Cell Sorter (Becton Dickinson). Erythroblasts population were differentiated as previously described^{17 (link)} into four populations: pro-erythroblasts (pro-E, Ter119^med CD71^high FSC^high), basophilic erythroblasts (Ter119^high CD71^high FSC^high), polychromic erythroblasts (Ter119^high CD71^high FSC^low) and orthochromic erythroblasts (Ter119^high CD71^low FSC^low).