Mitosox

Keratinocytes from perilesional vitiligo skin were seeded on glass cover slips and loaded with the mitochondrial superoxide-specific fluorescent probe MitoSOX (3 μM) and H₂DCFDA (2.5 μM; Invitrogen, Carlsbad, CA, USA) – dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v) – which was added to cell culture media for 15 min. at 37°C. Cells were fixed in 2.0% buffered paraformaldehyde for 10 min. at room temperature and the H₂DCFDA and MitoSOX fluorescence analysed with a Leica TCS SP5 confocal scanning microscope (Mannheim, Germany) equipped with an argon laser for fluorescence analysis. A series of optical sections (1024 × 1024 pixels) 1.0 μm in thickness was taken through the cell depth at intervals of 0.5 μm with a Leica 20× objective and then projected as a single composite image by superimposition. Mitochondrial superoxide and ROS generation were also monitored by flow cytometry: single-cell suspensions were incubated with MitoSOX (0.5 μM) and H₂DCFDA (1 μM; Invitrogen) for 15 min. at 37°C and immediately analysed with a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA).

H9c2 cells seeded on glass coverslips were loaded with the ROS-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen, CA, USA; 2.5 μmol L⁻¹) or the mitochondrial ${\mathrm{O}}_2^{\cdot -}$ -specific fluorescent probe MitoSOX (Invitrogen; 3 μmol L⁻¹) - dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v) – which were added to cell culture media for 15 min at 37 °C, as described^{18 (link)}. The cells were fixed in 2% buffered paraformaldehyde for 10 min at room temperature and the H₂DCFDA and MitoSOX fluorescence analysed using a Leica TCS SP5 confocal scanning microscope equipped with an argon laser source (excitation λ 488 nm or 543 nm, respectively) and a x63 oil immersion objective. ROS and mitochondrial ${\mathrm{O}}_2^{\cdot -}$ generation were also monitored by flow cytometry^{54 (link)}: briefly, single-cell suspensions were incubated with H₂DCFDA (1 μmol L⁻¹) or MitoSOX (0.5 μmol L⁻¹) for 15 min at 37 °C and immediately analysed using a FACSCanto flow cytometer (Becton–Dickinson). Data were analyzed using FACSDiva software (Becton–Dickinson).

Cells were stained with dihydroethidium (DHE, ThermoFisher Scientific) to measure intracellular peroxides or with MitoSOX (ThermoFisher Scientific) to assess mitochondrial superoxide. Cells were incubated with either 10 μM DHE or 5 μM MitoSOX for 30 min at 37 °C in the dark and analysed by flow cytometry or in a DMi8 fluorescence microscope (Leica).
Extracellular release of hydrogen peroxide was measured by Amplex Red assay (ThermoFisher Scientific) as described (Miwa et al., 2016 (link)) in a 96 well plate using a fluorescent plate reader (FLUOstar Omega, BMG Labtech) at excitation 544 nm and emission 590 nm at 37 °C.

HeLa cells were cultured on a Lab-Tek Chamber glass slide. Fluo-4-AM (5 μM), MitoSOX (5 μM), and DCF (5 μM) (Thermo Fisher, MA, USA) were added in HBSS buffer (0.49 mM MgCl₂, 0.41 mM MgSO₄, 5.33 mM KCl, 0.44 mM KH₂PO₄, 4.17 mM NaHCO₃, 137.93 mM NaCl, 0.34 mM Na₂HPO₄, 5.56 mM D-glucose, with or without 1.26 mM CaCl₂) (Gibco-Thermo Fisher, MA, USA) and incubated for 10 min. Afterward, the cells were washed once with HBSS, and peptides were treated with HBSS. Time-lapse images were obtained using an Argon laser scanning confocal microscope (Leica TCS SP5 Microsystems, Wetzlar, Germany) at 10-s intervals for 10 min at an excitation of 488 nm for Fluo-4 and MitoSOX, or at 496 nm for DCF. As mitochondrial markers, MitoTracker Green (0.5 μM) and MitoTracker Red (0.1 μM) were preincubated (green for 30 min and red for 2 min), anti-TOMM20 antibody (Abcam, Cambridge, UK) was used for immunocytochemistry and immunofluorescence (ICC/IF), and mito-DsRed2 were transfected with Effectene (Qiagen, Hilden, Germany) on the day before confocal microscopy. Other details are available in the manufacturer’s instructions.