Determination of IL1α, TNF-α, IL12, and IL10 Levels

Gastric tissue samples were ground and homogenized with ice-cold RIPA lysis buffer containing the protease inhibitor cocktail. The culture supernatant of the macrophages was collected and frozen at –20°C until measurement. According to the manufacturer's instructions, the cytokine levels of IL1α, TNF-α, IL12, and IL10 in the samples were determined using ELISA kits (Proteintech). Ninety-six–well plates were coated with the antibodies to IL1α, TNF-α, IL12, and IL10 at 4°C overnight. After blocking with buffer containing 2% FBS for 1 hour, the samples and standards were added at various dilutions and incubated at 37°C for 1 hour. After washing 3 times, the samples were incubated with the biotinylated detection antibody (diluted 1:1000) with buffer containing 1% bovine serum albumin at 37°C for 1 hour. After washing 3 times, the samples were incubated with horseradish peroxidase–labeled streptavidin antibody (diluted 1:5000) at 37°C for 1 hour. After the final washing, 100 μL 3, 3', 5, 5'-tetramethylbenzidine chromogenic substrate was added and reacted at 37°C for 20 minutes. Subsequently, 50 μL stop solution was added to each well and the absorbance (optical density) was measured at 450 nm using a microplate reader.