Human il 1β duoset elisa kit

The human IL-1β DuoSet ELISA kit was purchased from R&D Systems, Biomedica, Vienna, Austria. All other ELISA kits were purchased from Peprotech, Vienna, Austria. The assays were performed according to the manufacturers' instructions. Briefly, NUNC MaxiSorp flat-bottom 96-well plates (eBioscience, Affymetrix, Vienna, Austria) were coated with a capture antibody overnight and blocked with PBS containing 1% bovine serum albumin (BSA) for 1 h. After washing, cell supernatants were added and incubated for at least 2 h at room temperature (RT). Plates were washed again and the biotinylated detection antibody was added. After incubation for 1 h at RT, plates were again washed and avidin-conjugated horseradish peroxidase was added. After 30 min of incubation and another washing step, the substrate (TMB from Sigma-Aldrich) was added. The reaction was stopped by adding 2 M sulphuric acid. Colour intensity was measured at 450 nm and a reference wavelength of 650 nm was subtracted. The respective standards were used to calculate total protein concentrations.

Cytokines were quantified in supernatants using human IL-1β DuoSet ELISA kit (DY201, R&D systems), human IL-18 ELISA Kit (7620, MBL International), human IL-8 DuoSet ELISA kit (DY208, R&D systems), and human MCP-1 DuoSet ELISA kit (DY279, R&D systems), according to the manufacturers’ instructions.

IL-1β level in the supernatants of the control and treatment groups was evaluated by the human IL-1β DuoSet ELISA Kit (Cat No. DY201) (R&D Systems, Inc., Minneapolis, MN, USA) following the specific protocol instructed by the manufacturer. Briefly, the plate was coated with capture antibody at 4 °C for 24 h. Next, the plate was blocked at room temperature (RT) for 1 h and incubated with sample culture supernatants at RT for 2 h. Then, the detection antibody was added, and the plate was incubated (2 h). The plate was washed 3 times, and avidin-HRP solution was added (20 min). Finally, TMB substrate solution was added to the plate and color development was stopped by a stop solution after 20 min. The signal was measured at 450 and 570 nm with a plate reader (BioTek Instruments, Winooski, VT, USA).