Mitosox red mitochondrial superoxide indicator

ROS was measured by means of the 2′,7′ –dichlorofluorescin diacetate (DCFDA)–Cellular Reactive Oxygen Species Detection Assay Kit (ab113851, Abcam, Cambridge, UK), according to the manufacturer’s protocol. SIRC cells were plated into 96-well black plates at a density of 1 × 10⁴ cells/well for 24 h. After overnight growth, SIRC cells were grown in the control medium (CTRL), or in the presence of 10 nM NAP, or exposed to ultraviolet irradiation (UV-B) for 30 s with or without NAP for 24 h. Cells were washed gently in PBS twice and incubated with 25 μM DCFDA previously dissolved in a buffer solution for 45 min in the dark. The determination of the ROS concentration was performed by measuring the DCF fluorescence (λex = 495 nm, λem = 529 nm) with VarioskanTM. Twelve replicate wells were used for each group.
Mitochondrial-derived ROS (mtROS) detection was performed using the MitoSOX™ Red mitochondrial superoxide indicator (ThermoFisher Scientific, Milano, Italy). Cells were stained using 3 µM of the MitoSOX™ Red mitochondrial superoxide indicator (ThermoFisher Scientific, Milano, Italy). After incubation for 10 min at 37 °C, cells were washed three times and analyzed by MACSQuant Analyzer 10 (Miltenyi Biotec, Bologna, Italy).

Intracellular iron concentration was measured using the Iron Assay kit (Sigma-Aldrich, MAK025) according to the manufacturer’s instructions. Total intracytoplasmic ROS as well as specific mitochondrial superoxide species were assessed by flow cytometry using the fluorescent probes 2′-7′- Dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific) and MitoSOX™ Red Mitochondrial Superoxide Indicator, respectively. For the quantification of mitochondrial superoxide levels, cells were cultured for 72 h and then incubated with 2 mM MitoSOX™ Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific) for 30 min at 37 °C. Intracellular ROS levels were instead assayed by staining the cells with 1.5 mM CM-H2DCFDA (Thermo Fisher Scientific) for 30 min at 37 °C. Fluorescence intensity was measured by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorimeter (BD Biosciences, Bedford, MA, USA). The results were analyzed with FlowJo software (Tree Star, Inc., Williamson Way Ashland, OR 97520, USA). Three independent experiments were conducted.