Intracellular ROS and Mitochondrial Membrane Potential Assays

Fluorescent probes 2′,7′-Dichlorodihydrofluoresce in diacetate (Solarbio, China) were used to detect intracellular ROS in cells. DCFH-DA as oxidative sensitive fluorescent probe allowed to assess specific intracellular ROS by being oxidised to fluorescent DCF (36 (link)).The loss of MMP was estimated by JC-1 kit according to manufacturer’s introduction (Beyotime, China). To perform assays, HaCaT cells (2 × 10⁵ cells/well) were paved overnight. Then, the cells were pretreated with deucravacitinib or Deu@PEPS (0.5 μg/ml)and co-stimulated by TNF-α and IL-17A for 24 h. The cells were dyed with JC-1 dye or DCFH-DA probes (10 μM) in the sunblock and washed to remove remaining probes. The staining was tested using the fluorescence microscope.