Il 1α elisa kit

An IL-1α ELISA Kit (Quantikine, R&D Systems, Abingdon, UK) was used to measure IL-1α, we used, and followed the specified protocol. Assay diluent was added to 200 μL of test samples, which were then left for 2 hr at room temperature. After washing three times with washing buffer, we added 200 μL of IL-1α conjugate, and left at room temperature for 1 hr. After washing three times with washing buffer, 200 μL of TMB/H 2 O 2 solution was added, samples were shielded from light, and left for 20 min at room temperature. Subsequently, we added reaction stop solution (2N H 2 SO 4 ) and measured the absorbance at 450 and 540 nm within 30 min. Differences in absorbance were taken as the measured values, and the concentration of IL-1α was calculated using a prediction curve (calibration curve) prepared with standard samples.