Cells were isolated from tumors as described previously [26 (link)] and resuspended in media. A portion of cells was treated with 10 μg/ml of brefeldin-A (BFA) for 16 h in media alone or in media containing either 100 nM of GDC-0152 or 100 μg/ml of LPS. The remaining portion of untreated cells was used for cellular phenotyping. Cells were mixed with sorting buffer (PBS, 4% FBS, 5 mM EDTA) containing a cocktail of surface staining antibodies: CD49b(DX5)-PE, CD3-APC, Siglec-F-APC, F4/80-PE-Cy7, CD11c-V450, Ly6c-APC-Cy7, CD103-BV510 and Ly6G-BV711 (BD Biosciences) for 30 min at 4 °C, washed once with PBS and analyzed on a FACS ARIA III (BD Biosciences). mCherry fluorescence was used to identify tumor cells. For intracellular staining, samples treated with BFA were stained using the same antibody cocktail and then fixed with 1% paraformaldehyde for 15 min at room temperature in the dark. Samples were washed once with PBS and incubated with a TNFα-FITC antibody (BD Biosciences) in 0.4% saponin/PBS for 1 h at RT, washed and analyzed on a FACS ARIA III to detect TNFα positive cells co-stained with phenotyping markers. Flow cytometric data were analyzed using FCS Express (De novo Software; USA).
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