ELISA kit (ThermoFisher) was used to measure the secretion of interleukin (IL)-6 and IL-8). First, the ELISA 96-well plate was coated with 100 μL IL-6 and IL-8 capture antibody, and incubated overnight at 4C. The ELISA plate was washed with phosphate buffered saline (Wash Buffer) containing 0.05% Tween 20. Add 200μL ELISA/ELISPOT (1×) to each well and incubate for 1h. Then, 100 μL of sample was added to incubate for 2 hours, 100 ml of detection antibody was added to incubate for 1 hour, and 100 μL of streptavidin-horseradish peroxidase (HRP) were incubated for 30 minutes. Finally, add 100μL of tetramethylbenzidine (TMB) solution, incubate for 15min and add 50ul of stop solution to show orange yellow. After the end, use a microplate reader (BioTekEpoch2) to detect the absorbance at a wavelength of 450nm). The concentration of each sample was calculated according to the standard curve of IL-6 and IL-8.
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