Immunostaining of Erythroid Progenitors

Single-cell suspensions of freshly collected bone marrow cells were immunostained with APC-H7 (560185, BD Biosciences) or APCeFluor780 (eBioscience 47-1172-82) -conjugated rat anti-KIT, FITC-conjugated rat anti-CD71 (553266, BD Biosciences), APC-conjugated rat anti-Ter119 (557909, BD Biosciences) and PE-conjugated streptavidin (554061, BD Biosciences) and biotin-conjugated rat anti-CD123 (555070, BD Biosciences) antibodies for the detection of erythroid progenitor (CFU-E) cells, as previously described [52 (link)]; FITC-conjugated rat anti-CD11b (553310, BD Biosciences), PerCP-Cy^TM5.5-conjugated rat anti-mouse CD19 (45-0193-82, eBioscience) and PE-conjugated rat anti-mouse CD4 (553049, BD Biosciences), and APC-conjugated rat anti-myeloid CD8 (553035, BD Biosciences) and PE-Cy^TM7- conjugated rat anti-myeloid B220 (25-0452-82, eBioscience) antibodies were used for the detection of freshly collected bone marrow cells. Stained cells were analysed with a BD Accuri C6 or a Fortessa analyser (BD Biosciences), and FlowJo (Tree Star) was used for data acquisition and analysis.
Additional details of the methods are included in the supplementary data.