4.6. Measurement of Cytokines and Chemokines

HaCaT cells (5.0 × 10⁶ cells/mL) were seeded in a 12-well plate. For ELISA analysis, the cells were pre-treated with compounds for 3 h and then stimulated with TNF-α/IFN-γ (each for 5 ng/mL), with co-treatment for 24 h. After that, the cell culture supernatant was collected and IL-6, IL-8, RANTES, and MDC levels were evaluated using an ELISA kit, according to the manufacturer’s instructions. For experiments of compound 1 with Snpp, firstly, HaCaT cells were treated with Snpp (40 μM) for 2 h, and then the compound 1 was added into the culture media for another 3 h, Finally, we stimulated TNF-α/IFN-γ with HaCaT cell for 24 h. After that, the cell culture supernatant was collected and IL-6, IL-8, and MDC were detected using an ELISA kit (Biolegend, San Diego, CA, USA).