Ls macs cell separation columns

Primary monocyte-derived DCs were differentiated from CD14⁺ peripheral blood monocytes, and matured with LPS(100ng mL⁻¹) for 2 days prior to use.^{10 (link)} Primary human CD4⁺ T cells were positively isolated from CD14-depleted peripheral blood mononuclear cells (PBMCs) using CD4-conjugated magnetic beads and LS MACS cell separation columns (Miltenyi Biotech). Positively isolated CD4⁺ T cells were activated with 2% PHA (Invitrogen) for 2 days, washed and cultured in IL-2 (50 U/mL) containing RPMI supplemented with 10% FBS.

Human PBMCs were isolated from leukoreduction filters (Haemonetics) obtained from healthy donors by density gradient centrifugation. Primary CD14+ monocytes were isolated from PBMCs using mouse anti-human CD14 monoclonal antibody-conjugated magnetic beads and LS MACS cell separation columns (Miltenyi Biotec) according to the manufacturer’s protocol. Primary monocyte-derived macrophages (MDMs) were generated by culturing CD14+ monocytes in RPMI-1640 with 10% human AB serum, 10% FBS, 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 0.29 mg/ml L-glutamine (Gibco) for six days to differentiate into MDMs. Following differentiation, MDMs were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.29 mg/ml L-glutamine. The genetic sex of a subset of the donors was determined by PCR amplification of the SRY gene located on the Y chromosome. PM1 cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.29 mg/ml L-glutamine. 293T cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.29 mg/ml L-glutamine.