E el m0048c

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).

The colon tissues were homogenized in PBS on ice and then centrifugated at 500 g for 10 min at 4 °C and the supernatant was collected and analyzed. The cell culture medium was centrifugated at 1000 g for 20 min at 4 °C to remove impurities and cell debris, and the supernatant was collected and analyzed. The levels of TNF-α (E-EL-M3063; Elabscience, Houston, TX, USA), IFN-γ (E-EL-M0048c; Elabscience), IL-6 (E-EL-M0044c; Elabscience), IL-1β (E-EL-M0037c; Elabscience), IL-18 (E-EL-M0730c; Elabscience), and IL-17 (BMS6001TEN; Invitrogen) were determined using commercial ELISA kits. The optical density was measured at 450 nm using a microplate reader.