Determination of TNF-α, IL-1, and IL-6 release in microglia

Effects of RBE and α-tocopherol were also studied by determining the release of various cytokines. Briefly, microglia were pre-incubated either with RBE (50–300 μg/ml) or with α-tocopherol (10–100 μM). Afterwards, LPS (10 ng/ml) was added for 24 h, and release of tumor necrosis factor (TNF)-α and IL-6 was determined in the cell supernatants which were collected after centrifugation at 1000g for 5 min at 4 °C. For the release of IL-1β at the end of the incubation, ATP (1 mM) was added for 30 min in all the wells followed by the collection of cell supernatants. Addition of ATP was important since maturation and release of IL-1β is dependent on the IL-1β converting enzyme (ICE)/caspase 1. For determination of TNF-α (eBioscience, Frankfurt, Germany), IL-6 (Thermo Fisher Scientific, Darmstadt, Germany) and IL-1β (R&D Systems Europe, Ltd., Abingdon, UK), commercially available ELISA kits were used. All the measurements were done at 450 nm according to the manufacturer’s instructions. For TNF-α, standards from 16 to 2000 pg/ml were used with sensitivity 16 pg/ml. IL-6 has a range between 23.5 and 1500 pg⁄ml with sensitivity <5 pg/ml, and IL-1β standards were between 31.5 and 2000 pg/ml with sensitivity of <5 pg/ml.