Elx800 uv vis microplate reader

Morning blood samples (5 ml) were drawn from all patients by venepuncture in plain vacutainers at the same time (8:00 a.m.) following an eight‐hour fast. The vacutainers were then centrifuged at 4500×g for 15 min to obtain separated serum samples, which were transferred to Eppendorf tubes and maintained in a deep freezer at −80℃ until analysis. Serum levels of CREB1, BDNF, 5‐HT, TNF–α, NF‐ κB, and FAM19A5 were measured using specific commercial ELISA kits purchased from MyBioSource, Inc. (USA). The measurements were carried out according to the manufacturers’ instructions using Biotek ELx800 UV‐Vis Microplate Reader (USA).

Murine glioma cell supernatants were applied to the ELISA plate pre-coated with monoclonal antibodies specific for mouse TGF-β2 (R&D Systems Quantikine ELISA). An enzyme-linked polyclonal antibody, the TGF-β2 conjugate, was then applied creating a quantitative sandwich enzyme immunoassay. Once the Substrate Solution and consecutive Stop Solution were added to each well, an enzyme reaction yielded a color change with varying intensities. The color intensity of each corresponded to a specific amount of TGF-β2 analyzed by Bio-Tek ELx800 UV–Vis microplate reader which calculated optical density. A standard curve was constructed by plotting mean absorbance for each lane of standard, and therefore allowed for the calculation of concentration in pg/mL of TGF-β2. Each sample was assayed in technical quadruplet (n = 4), and the experiment was performed three times with little variability between results.