Il 6 and tnf α elisa kits

CSE (25, 50 and 100 μg/mL) was treated for 1 h to BV-2 microglia cells (1 x 10⁵ cells/well) cultured on 96 well plates and stimulated with LPS (1 μg/mL) for 4 h. The supernatants removed were evaluated for TNF-α and IL-6 levels using TNF-α and IL-6 ELISA kits according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA).

HPLC grade methanol, acetonitrile, formic acid, Folin-Ciocalteu reagent and dimethylcinnamaldehyde were purchased from Sigma-Aldrich (Oakville, ON, Canada). The liquid chromatography standards were purchased as follows: Cyanidin-3-O-glucoside from Extrasynthese (Genay Cedex, France); phloridzin, phloretin, chlorogenic acid, ferulic acid and caffeic acid from Sigma-Aldrich; catechin, epicatechin, quercetin, quercetin-3-O-galactoside and quercitin-3-O-glucoside from ChromaDex, Inc. (Santa Ana, CA, USA); quercitin-3-O-rhamnoside, quercitin-3-O-galactoside and anthocyanin standards from Indofine Chemical Company (Hillsborough, NJ, USA). Hydrochloric acid, sulfuric acid, and 96-well microplates were purchased from Fisher Scientific (Ottawa, ON, Canada). Phorbol 12-myristate 13-acetate, diclofenac sodium salt, nimesulide and lipopolysaccharide were obtained from Sigma-Aldrich. COX-2 human ELISA kit was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Prostaglandin E₂ EIA kit and nitric oxide quantification kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The TNF-α and IL-6 ELISA kits were purchased from BD Biosciences (San Diego, CA, USA).

Commercially available IL-6 and TNF-α ELISA kits (BD Biosciences) were used to determine the IL-6 and TNF-α concentrations in the supernatant of RAW264.7 cells according to the manufacturer’s protocols.
Splenocytes from BALB/c mice were plated in 96-well plates at a density of 2.5 × 10⁶ cells/well. Supernatants from the cultured splenocytes were measured using a cytometric bead array (CBA) immunoassay with a mouse T helper type 1 (Th1)/type 2 (Th2)/type 17 (Th17) cytokine kit according to the manufacturer’s guidelines (BD Biosciences). The cytokine levels in the culture supernatants were detected using CBA mouse flex set capture beads, which consisted of a population of single beads with a distinct fluorescence intensity and a coating of specific antibodies recognizing detectable cytokines. The captured cytokines were detected with specific antibodies conjugated to PE dye. The samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences) with FCAP array multiplex analysis software to calculate the cytokine concentrations and plot the standard curves.