Facsgallius flow cytometer

For cell cycle analyses, BrdU incorporation was done ex vivo as previously described (Malik et al., 2013 (link)). Briefly, E9.5 circulating erythroblasts were cultured for 90 min in erythroid differentiation media (IMDM, 20% FBS (Biological Industries Inc., Israel), 10% PFHM-II (Invitrogen), 2 mM L- glutamine, 150 μM MTG (Sigma), and 1 U/mL recombinant human EPO (PeproTech Asia), supplemented with 10 μM bromodeoxyuridine (BrdU). BrdU incorporation was detected using a FITC BrdU flow kit (BD Biosciences), according to the manufacturer’s instructions, on a FACSGallius flow cytometer (Beckman Coulter). Data were acquired and analyzed using Kaluza^® for Gallios Acquisition Software (Beckman Coulter).

To monitor erythroid differentiation status, circulating E9.5–E11.5 erythroblasts were stained with PE-conjugated anti-Ter119 and APC-conjugated anti-CD71 (Biolegend). Antibodies were used at 1:200. Dead cells were excluded with 7-AAD staining. For analyses of apoptosis, erythroblasts were co-stained with Ter119, CD71, 7AAD and Annexin V (Biolegend). All the experiments were performed on a FACSGallius flow cytometer (Beckman Coulter). Data were acquired and analyzed using Kaluza^® for Gallios Acquisition Software (Beckman Coulter). Values represent averages across biological replicates ± SEM. The student’s paired t-test was used to determine significance (p < 0.05).