Quantifying Cytokine Levels in Pancreatic Cancer

Equal numbers (1 × 10⁶) of cells CD18/HPAF-scram (control), CD18/HPAF-shKRAS, HPNE, HPNE-KRAS, E6-E7-st, E6-E7-st-KRAS, E6-E7-st-KRAS-NSC, E6-E7-st KRAS-shCXCR2 and KRAS-PDAC cells were plated in 60 mm dishes in complete medium. After attachment of cells to the plate, the medium was changed to serum-free DMEM. Supernatants of cultured cells were collected at 24 hours or 72 hours. Protein was isolated from tumors by homogenizing in a bullet blender using Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) as a lysis buffer. ELISA assays for hCXCL8 and hCXCL1 were performed as described previously [39 (link)]. hCXCL5, mCXCL2, mCXCL5 and mCXCL7 ELISAs were performed using a duoset kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. All the experiments were performed in duplicates.