Il 1β eia kit

ELISA assays were performed to determine the amount of IL-1β in the kidney. Rats were anesthetized with ketamine/xylazine (25:2.5 mg/kg of body weight, i.p.). Afterwards the kidney was removed and homogenized with an Ultra-Turrax homogenizer in buffer (ratio: 0.1 g renal tissue: 1 ml lysis buffer) containing 100 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% SDS, 1 μM PMSF and a protease inhibitor cocktail (Halt™ Protease Inhibitor Cocktail, Pierce, Rockford, IL, USA). Then, samples were centrifuged at 14,000× g for 10 min (Eppendorf 5415C, Eppendorf, Hamburg, Germany). Supernatants were collected and protein content assayed by Bicinchoninic Acid (BCA) Protein Assay. Cytokine levels were determined by sandwich ELISA, according to the manufacturer’s protocol (IL-1β EIA kit, Enzo Life Science, USA). For the assay, 100 µL of samples were added per well and incubated at 4 °C overnight. A calibration curve with recombinant cytokine was included. Samples were incubated with secondary antibody at room temperature for 2 h and the reaction was developed with avidin–HRP and substrate solution. Absorbance was measured at 450 nm with reference to 570 nm with the microplate reader Synergy HT (Biotek Instruments). The results were normalized by protein amount in pg/g protein.

IL-1β was determined in the extracellular medium under different conditions in mesangial cells. Samples were centrifuged at 14.000 g for 40 min. Supernatants were collected and protein content was assayed using the BCA method. IL-1β levels were determined by sandwich ELISA, according to the manufacturer’s protocol (IL-1β EIA kit, Enzo Life Science, Farmingdale, NY, United States). For the assay, 100 µL of samples were added per ELISA plate well and incubated at 4°C overnight. A calibration curve with recombinant cytokine was included. Detection antibody was incubated at room temperature for 1 h and the reaction developed with avidin–HRP and substrate solution. Absorbance was measured at 450 nm with reference to 570 nm with the microplate reader Synergy HT (Biotek Instruments).