Immunomodulatory Effect after Irreversible Electroporation in Patients with Locally Advanced Pancreatic Cancer

He, Chaobin; Wang, Jun; Sun, Shuxin; Zhang, Yu; Li, Shengping

. . Assays of Immune Parameters. The quantitative sandwich enzyme immunoassay technique (ELISA kit, R&D system, Minneapolis, MN) was adopted to measure serum concentrations of cytokines, including IL-2, IL6, IL-10, interferon-(IFN-), and tumor-necrosis factor (TNF). During the procedure of measure, 50 to 100 l of assay diluent was added to the 96-well polystyrene microplate, which was precoated with murine monoclonal antibody against IL-2, IL-6, IL-10, IFN-, and TNF. Serum samples were incubated at 37 ∘ C for 2 hours and then the plates were aspired and washed three times. Same incubation was repeated after 200 micoliters of conjugate was added. Then, plates were incubated at 37 ∘ C for 20 to 30 minutes after 200 l of substrate solution was added. Finally, 50 l of stop solution was added to the plates. A microplate reader (ClinicalBio 128c, Austria) was used to read optimal density (OD) within 30 minutes at 450 nm wavelength, whose references were set to 550 and 620 nm.