Kaluza for gallios acquisition software

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).

The following mAb were used: CD3 krome orange [clone UCHT1] (Beckmann Coulter, Brea, CA, USA), CD4 phycoerythrin-cyanin 7 (PE-Cy7) [clone SK3] (BD Bioscience, Franklin Lakes, NJ, USA), CD8 pacific blue [clone B9.11] (Beckmann Coulter), CD11c PE [clone S-HCL3] (BD Bioscience), CD14 fluorescein isothiocyanate (FITC) [clone MϕP9] (BD Bioscience), CD19 allophycocyanin [clone HIB19] (BD Bioscience), CD137 allophycocyanin [clone 4B4-1] (BD Bioscience), CD83 PE [clone HB15] (Miltenyi Biotec), HLA-DP PE [clone B7/21] (21 (link)) (Leinco Technologies, Inc. St.Louis, MO, USA), HLA-DR PE [clone L243] (BD Bioscience), murine IgG2A PE [clone S43.10] (Miltenyi Biotec) and murine IgG3 PE [clone A112-3] (BD Bioscience). Flow cytometric determinations were performed on a Gallios™ 10/3 cytometer (Beckman Coulter, Brea, CA, USA), using the Kaluza for Gallios Acquisition software (Version 1.0, Beckman Coulter) and the data were analyzed with Kaluza Analysis Software (Version 1.3, Beckman Coulter).