Opteia mouse tnf α elisa set

Type I interferon secretion was determined by an ISRE-luciferase bioassay [67 (link)]. L929 ISRE-luciferase cells were plated the day before at 10⁵ cells per well in a 96 wells-plate. Supernatants from infected BMDMs were added for 4 h onto the ISRE-luciferase cells. Luciferase luminescence was detected using Bright Glo Assay (Promega) following the manufacturer’s instructions. TNF-α secretion of J774 cells upon 2 h of LPS stimulation was performed according to our previously established protocols [7 (link)], using the BD OptEIA Mouse TNF-α ELISA Set (BD Biosciences) according to the manufacturer’s instructions.

Frozen livers were homogenized (0.2 g tissue/mL) in lysis buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate, 150 mM NaCl; 1 mM EDTA, 17.5 μg/mL aprotinin; 5 μg/mL bestatin, 10 μg/mL leupeptin, 20 μg/mL E-64, 1 mM Na₃VO₄, 10 mM Na₄P₂O₇) [14 (link)]. One tablet of complete cocktail inhibitor (Roche, Indianapolis, Indiana, USA) was added for each 50 mL buffer. Samples were normalized with respect to the total protein concentrations using the Bio-Rad Protein Assay kit II (cat. 500-0002) before quantification of cytokines. The cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using the following kits: TNF-α (BD OptEIA Mouse TNF-α ELISA Set, cat. 555268), IL-6 (BD OptEIA Mouse IL-6 ELISA Set, cat. 555240), IL-12p70 (BD OptEIA Mouse IL-12p70 ELISA Set, cat. 555256), and IL-17A (eBioscience Mouse ELISA Ready-SET-Go, cat. 88-7371-88).