Measurement of expression of cytokines

The concentration of IL-6, IL-10, IL-17 and TGF-β were measured. After diluting the antibody magnetic beads 1:50, 50 µl of the sample was added to each well in a 96-well plate and the plate was placed on a magnetic rack. After 2 min, the supernatant was discarded and washed with wash buffer once. Additional care was taken not to detach the 96-well plate from the magnetic plate rack during the process to prevent the magnetic beads from falling off. The provided reagents in each kit were dissolved according to the manufacturer's protocol and different dilutions were added to the 96-well plates. DMEM/F12 complete medium was added to the final well as the blank control group. Experiments where performed with 6 repeats per each condition and 2 groups were used to plot the standard curve. A total of 50 µl of sample was added to the remaining wells and 25 µl of wash buffer was added to these wells. The plate was incubated for 2 h at room temperature to allow the magnetic beads to bind to the corresponding cytokines. After incubation, the wells were washed with the special solution three times and 25 µl of a diluted solution of the corresponding cytokine antibody was added, incubated at room temperature in the dark for 30 min, rinsed with special solution, 50 µl of phycoerythrin dye was added and incubated on an oscillator for 30 min at room temperature. After the incubation, the buffer was cleaned once with the special solution and 120 µl reading buffer was added to each well. The buffer was placed on the oscillator for 5 min to ensure the solution was adequately mixed prior to taking measurements.