BM-derived myeloid progenitor cells were generated from hematopoietic progenitor cells isolated from BM using a Miltenyi MACS lineage depletion kit. Cells were stimulated for 3 d in IL-3 (25 ng/ml), IL-6 (25 ng/ml), and SCF (50 ng/ml), washed, and stimulated for indicated times with rIFN-α4 (2,000 U/ml, PBL Assay Science). BMDMs were differentiated by incubating total BM cells in 15-cm tissue culture plates with recombinant M-CSF (100 ng/ml; Genentech) in DMEM for 7-10 d. M-CSF (100 ng/ml) was replenished every 3 d during culturing. BMDMs were plated overnight in M-CSF, rested for 4 h and stimulated for the indicated times with rIFN-α4 (2,000 U/ml; PBL Assay Science). For cell transfection experiments, 3T3 cells were transfected using Lipofectamine LTX with plus reagent (Life Technologies) according to manufacturer’s instruction.
Holmes D.A., Suto E., Lee W.P., Ou Q., Gong Q., Smith H.R., Caplazi P, & Chan A.C. (2015). Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling. The Journal of Experimental Medicine, 212(7), 1081-1093.
+ Open protocol