Human il 17a high sensitivity elisa

Freshly isolated PBMCs were seeded in a 48–well plate (1 × 10⁶ cells in 0.5 mL of the culture medium per well) and treated with the tested compound at 5 µM concentrations for 30 min and then, 10 ng/mL IL–15 (R&D Systems, Minneapolis, MN, USA) was added for 48 h. Next, the culture media from each well were collected, centrifuged (10,000× g, 10 min, 4 °C), and frozen at −80 °C until the level of cytokine was measured using ELISA tests. Cells were harvested, lysed in 0.1 M NaOH, and frozen at −80 °C until the total protein level measurement. Each experiment was performed using cells isolated from three blood donors, each in two technical replicates.
Total protein levels were measured in PBMCs using the BCA Protein Assay Kit (Pierce Biotechnology, Waltham, MA, USA). The concentrations of IL–17 and TNF–α were measured in culture supernatants of cytokine–stimulated cells using the Human TNF–α Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and Human IL–17A High Sensitivity ELISA (eBioscience, San Diego, CA, USA), respectively, according to the manufacturer’s instructions. The values obtained for IL–17 and TNF–α concentrations were calculated per 1 mg of total protein.