EROD assay

CYP1A1 enzymatic activity was measured in mouse tail epidermal sheet biopsies and in vitro skewed Th17 cells by the EROD assay, in which the 7-ethoxyresorufin (Sigma-Aldrich) is converted to resorufin (Sigma-Aldrich). Tail mouse skin was processed by the mechanical removal of subcutaneous fat tissue and floated on 0.5% w/v trypsin (Sigma-Aldrich) for 1 hour at 37 °C. Epidermal sheets were mechanically separated, and quadruplicate from each epidermal sheet was transferred to a 96-wells plate. EROD reaction was initiated by adding 2 μM 7-ethoxyresorufin in sodium phosphate buffer (50 mM, PH 8.0) to each well. The plate was incubated at 37 ^oC for 20 minutes, the reaction was stopped by adding fluorescamine (150 μg/ml in acetonitrile), and epidermal sheets biopsies were discarded. Resorufin formation was measured against a resorufin standard curve on a plate reader (FLUOstar Omega, BMG LabTech, Ortenberg, Germany) with excitation and emission wavelengths of 535 and 590, respectively. CYP1A1 enzymatic activity was normalized for the skin protein content simultaneously determined by fluorescamine fluorescence of a standard curve of BSA (Sigma-Aldrich) at excitation and emission wavelengths of 390 and 480, respectively. In vitro skewed Th17 cells were assayed similarly, and CYP1A1 enzymatic activity was normalized to the IL-17 secreted in each well.