Human granulocyte macrophage colony stimulating factor

Primary human monocytes were isolated and pooled from buffy coats of four healthy blood donors (New York Blood Service, Long Island, NY) by positive selection using MACS CD14 microbeads (Miltenyi Biotec) as described in our previous study (33 (link)). The monocytes were differentiated into MDMs in the presence of 5 ng/ml human granulocyte-macrophage colony-stimulating factor (Miltenyi Biotec) for 7 days prior to respective experiments.

Primary human monocytes were isolated from peripheral blood mononuclear cells of four healthy donors, which were purchased from the New York Blood Service, using the MACS CD14 microbeads (Miltenyi Biotec) as described previously (78 ). The pooled monocytes from five donors were differentiated for 7 days into MDMs in the presence of 5 ng/ml human granulocyte–macrophage colony-stimulating factor (Miltenyi Biotec). Separately, 293T and THP-1 cells (42 (link)) transduced with LentiCRISPR empty vector control (SAMHD1 WT) or vector containing specific guide RNA targeting the SAMHD1 gene (SAMHD1 KO) were cultured in Dulbecco's modified Eagle's medium (DMEM) and RPMI, respectively, which were supplemented with 10% fetal bovine serum (FBS), penicillin–streptomycin (100 U/ml), and puromycin (1 μg/ml) at 37 °C, 5% CO₂. Separately, 293T cells that have been LentiCRISPR–Cas9 KO for RNaseH2 gene expression (RNaseH2 KO) were cultured under the same conditions as SAMHD1 KO 293T cells. The monocytic THP-1 cells were differentiated into macrophage-like nondividing cells, via treatment with 100 ng/ml phorbol 12-myristate 13-acetate for 72 h. On the other hand, Huh-7 and Vero E6 cells (American Type Culture Collection) were grown and maintained in DMEM and minimal essential medium containing 10% FBS and penicillin–streptomycin (100 U/ml) at 37 °C, 5% CO₂, respectively.