Elisa max deluxe set mouse il 6

About 1 × 10⁵ RAW 264.7 cells, suspended in DMEMg supplemented with NEAA, sodium pyruvate and 10% FBS, were seeded in each well of 96-well plates. After overnight incubation at 37 °C and 5% CO₂, the medium was removed and replaced with fresh medium plus 10% FBS containing 20 ng/mL LPS derived from P. aeruginosa (Sigma-Aldrich, St. Luis, MO) or each Esc peptide at different concentration or LPS in combination with each peptide. Samples were incubated at 37 °C and 5% CO₂ for 4 h. At the end of the treatment, the supernatants were collected and IL-6 concentration was evaluated by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s protocol (Mouse IL-6 ELISA MAX Deluxe set, Biolegend, San Diego, CA, USA). Cells stimulated with LPS alone and untreated cells served as controls.

Active TGF-β1 expression was measured using an ELISA kit (Boster, Wuhan, China), according to the manufacturer’s instructions. After the reaction, the optical density (OD) was measured at 450 nm by a microplate reader (Roche Molecular Diagnostics, Light Cycler 480II, Carlsbad, CA, United States).
In addition, collagen expression was measured with a Sirius Red Total Collagen Detection Kit (Chondex, #9062, Redmond, WA, United States), according to the manufacturer’s instructions, using 1 mg of tissue per mouse. The optical density was measured at a wavelength of 500 nm.
Tumor necrosis factor (TNF)-α and IL-6 levels in BALF were determined using the appropriate mouse ELISA kits (Mouse TNF-α ELISA MAX Deluxe Set and Mouse IL-6 ELISA MAX Deluxe Set, BioLegend, San Diego, CA, United States).

Serum sample were taken at 0, 4, 8, 18, after 20 mg/kg LPS injection. Cytokine array was performed with LEGENDplex Mouse inflammation Panel (Biolegend) according to the manufacturer’s protocol. Data analysis was proceeded by using LEGENDplex Data Analysis Software. Mouse serum levels of IFN-γ, IL-1α, and IL-6 were quantified with Mouse IFN-γ ELISA MAX Deluxe Set (Biolegend), Mouse IL-1α ELISA MAX Deluxe Set (Biolegend), and Mouse IL-6 ELISA MAX Deluxe Set (Biolegend) according to the manufacturer’s protocol. Mouse serum, BMDMs supernatant, and intracellular IL-12p70 were quantified by using ELISA MAX Deluxe Set Mouse IL-12 (p70; Biolegend).