T cells, B cells, Mph and DCs were sorted by flow cytometry using markers TCRβ, CD4, CD8, CD11c, CD11b, CD19, CD45R, MHC II as described before (12 (link)). Purity of sorted cells was generally >98% and viability was >97% as determined by 7-AAD staining.
Erythrocytes, normoblasts, reticulocytes and red blood cells were generated using a mouse adapted protocol for the long-term ex vivo erythroid differentiation culture protocol described by Giarratana et al (17 (link)) and Konstantinidis et al (18 (link)). Briefly, low-density bone marrow cells were cultured in erythroblast growth medium (StemPro-34 with 2.6% StemPro-34 supplement; Invitrogen), 20% BIT 9500 (StemCell Technologies), 900 ng/mL ferrous sulfate, 90 ng/mL ferrous nitrate, 10−6M hydrocortisone, penicillin/streptomycin, L-glutamine), in 3 subsequent phases. For the proliferative phase (days 1–5) cells were expanded with 100 ng/mL SCF, 5 ng/mL IL-3, and 2 IU/mL human erythropoietin (Amgen). In the differentiation step (days 6–7), the cells were supplemented with only erythropoietin in fibronectin coated plates. For enucleation (day 8–9) cells were grown without cytokines. Cells were sorted based on size gating and nucleotide staining using different combinations of Syto-16, Draq5, Mitotracker Red and MitotrackerGreen (Invitrogen).
Erythrocytes, normoblasts, reticulocytes and red blood cells were generated using a mouse adapted protocol for the long-term ex vivo erythroid differentiation culture protocol described by Giarratana et al (17 (link)) and Konstantinidis et al (18 (link)). Briefly, low-density bone marrow cells were cultured in erythroblast growth medium (StemPro-34 with 2.6% StemPro-34 supplement; Invitrogen), 20% BIT 9500 (StemCell Technologies), 900 ng/mL ferrous sulfate, 90 ng/mL ferrous nitrate, 10−6M hydrocortisone, penicillin/streptomycin, L-glutamine), in 3 subsequent phases. For the proliferative phase (days 1–5) cells were expanded with 100 ng/mL SCF, 5 ng/mL IL-3, and 2 IU/mL human erythropoietin (Amgen). In the differentiation step (days 6–7), the cells were supplemented with only erythropoietin in fibronectin coated plates. For enucleation (day 8–9) cells were grown without cytokines. Cells were sorted based on size gating and nucleotide staining using different combinations of Syto-16, Draq5, Mitotracker Red and MitotrackerGreen (Invitrogen).
