Recombinant human ifn α

Human lung adenocarcinoma cells (A549), human immortalized keratinocytes (HaCaTs), mouse macrophage RAW 264.7 cells and Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MDCK and mouse macrophage RAW 264.7 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Normal human dermal fibroblasts (HDFs) were purchased from Lonza, Basel, Switzerland, and grown in fibroblast basal medium (FBM) supplemented with FGM SingleQuots (Lonza). THP-1 human leukemia monocytes were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin and differentiated into macrophages in the presence of 100 ng/mL PMA (Sigma-Aldrich, St. Louis, MS, USA).
Recombinant human IFN-α, λ1, and λ2 were purchased from R & D Systems (Minneapolis, MI, USA), and IFN-β protein was obtained from PBL Assay Science (Piscataway, NJ, USA). Human IFN neutralizing monoclonal antibody and murine recombinant IFN-β protein were obtained from R & D Systems.

GBM1 cells were cultured overnight at 1x10⁵ cells/well. Cells were then treated with 100 pg of recombinant human IFN-α (R&D Systems), IFN-β (PBL Interferon Source), or IFN-γ (Peprotech) alone or in combination. After 24 hrs, cells were harvested and stained with anti-PD-L1-PE as above.