E el m0043c

Cytokine levels in the co-culture supernatant were measured by commercial ELISA kits. Mice were tested for IFN-γ (USCN, catalog No. SEA049Mu, China), IL-4 (Elabscience, catalog No. E-EL-M0043c, China), IL-17a (Elabscience, catalog No. E-EL-M0043c, China), TGF-β1 (USCN, catalog No. SEA124Mu, China), and IL-10 (USCN, catalog No. SEA056Mu, China) in accordance with the manufacturer's instructions.

Renal tissues levels of mouse IL-17A, IFN-γ (MULTISCIENCES: EK217/2-AW1, EK280/3-AW1), IL-6, IL-4 and IL-10 (Elabscience: E-EL-M0044c, E-EL-M0043c, E-EL-M0046c) were quantified by using an ELISA method according to the instructions. Renal tissue lysates were extracted according to the following formula: 50 mmol/L Tris–HCl, 0.2% Triton X-100, 2 mmol/L EDTA, 150 mol/LNaCl, 2 mmol/L EGTA, 0.3%IGEPAL, 10 μl/ml proteinase inhibitors cocktail, 10 μl/ml PMSF, and 10 μl/ml orthovanadate (21 (link)).

Mouse IL-4, IL-5, and IFN-γ ELISA Kit were commercially purchased from Elabscience (E-EL-M0043c, E-EL-M0722c, E-EL-M0048c, Wuhan, China). After seeding the antibody in the well for 12 hours, the reagent was replaced with 0.1 mL testing sample or standards, followed by incubation at 37°C for 60 minutes. Then, 0.1 mL of fresh horseradish peroxidase (HRP)-conjugated antibody was introduced into each reaction well, followed by a 60-minute incubation period. 0.1 mL of the temporarily prepared 3,3′,5,5′-Tetramethylbenzidine (TMB) reagent was then introduced and cultured for 30 minutes before introducing sulfuric acid to end the reaction. The optical density at a wavelength of 450 nm was measured using a microplate reader (DeTie, China).