2.4 ELISA
The expression of CXCL8 and IL-6 in the supernatant of the cell culture and other inflammatory factors, including IL-1, IL-6, IL-17A, and IL-23 in the serum and skin tissues of mice were detected by ELISA. ELISA plates were washed with PBS containing Tween 20 (0.05%) and blocked using assay diluents for 1 h at 37 °C. After washing, samples were serially diluted using assay diluents and added to the plates and then incubated for 2 h followed by the addition of diluted detection antibody at 37 °C. The plates were washed again; avidin-horseradish peroxidase (HRP) was added to the plates and incubated for 30 min. Next, the substrate solution tetramethylbenzidine (TMB) was added and incubated for 15 min; then, the reaction was stopped by adding 100 μL of acid stop solution. Absorbance was read at 450 nm within 20 min using an ELISA reader (Bio-Rad Laboratories, Hercules, CA).