Monocytes isolated from PBMCs using human CD14 positive selection kit (Stemcell) were cultured in X-Vivo-15 media (Lonza) with 2% human AB serum (Invitrogen), 1000 IU/mL human GM-CSF and 500 IU/mL human IL-4 (Peprotech) for 6 days. Fresh culture media containing GM-CSF and IL-4 were added on day 3. The resulting monocyte-derived dendritic cells (mDCs) were collected and seeded at 1 × 106 cells/well in 12-well plates. Cells were activated with 0.5 μg/mL of HA-tagged CD40L (R&D systems) and 2 μg/ml of anti-HA antibody (R&D systems); and treated with selumetinib or DMSO control for a further 2 days.
2 × 105 CT26 cells were cultured overnight prior to treatment with selumetinib or DMSO control for 2 days.
For flow cytometry analysis, mDCs were stained with fixable LIVE/DEAD violet (Life Technologies) and incubated with human TruStain FcX (Biolegend) before addition of: CD80-FITC (clone L307.4); CD83-PE (clone HB15e); CD86-APC (clone FUN-1) (BD Biosciences); HLA-DR-PE (clone L243, eBioscience). CT26 cells were stained with a viability stain, H2-Kd-PE (clone SF1–1.1) and PD-L1-APC (clone 10F.9G2, Biolegend). Stained cells were analyzed using a FACScantoII (BD Biosciences).
2 × 105 CT26 cells were cultured overnight prior to treatment with selumetinib or DMSO control for 2 days.
For flow cytometry analysis, mDCs were stained with fixable LIVE/DEAD violet (Life Technologies) and incubated with human TruStain FcX (Biolegend) before addition of: CD80-FITC (clone L307.4); CD83-PE (clone HB15e); CD86-APC (clone FUN-1) (BD Biosciences); HLA-DR-PE (clone L243, eBioscience). CT26 cells were stained with a viability stain, H2-Kd-PE (clone SF1–1.1) and PD-L1-APC (clone 10F.9G2, Biolegend). Stained cells were analyzed using a FACScantoII (BD Biosciences).