Il 35

The levels of cytokines (IL-4, IL-5, IL-10, and IL-35) in the culture supernatants were measured using sandwich ELISA. Plates were coated with anti-mouse IL-4, IL-5, and IL-10 (PeproTech, Rocky Hill, NJ, USA), or IL-35 (BioLegend, San Diego, CA, USA). After addition of the culture supernatants, the plates were incubated with biotinylated anti-mouse IL-4, IL-5, and IL-10 (PeproTech), or IL-35 (BioLegend) antibodies. Standard curves were generated using recombinant cytokines. The detection limits of these cytokines were 10–20 pg/mL.

Medium was removed, and new medium containing cytokines was used to stimulate keratinocytes for a variety of time points. Unless stated otherwise, IL-6, IL-23, IL-27, IL-35 (all from BioLegend), and IL-12 (Tonbo) were added to 1 to 2 ml of medium at a concentration of 100 ng/ml; IFN-α (BioLegend) or IFN-γ (PeproTech) was added to 1 to 2 ml of media at a concentration of 50 U/ml. For the cytotoxicity study, cells were suspended in CellTiter 96 AQueous One Solution Reagent and then incubated at 37°C for 1 to 4 hours in a humidified, 5% CO₂ atmosphere. Absorbance was read at 490 nm using a 24-well plate reader.