Mitosox

Cells were incubated with 2 μg/mL Hoechst 33342 (Thermo Fisher Scientific, H1399) and 3 μM MitoSOX^TM (Thermo Fisher Scientific, M36008) in DMEM supplemented with 5% (v/v) FCS, 1x penicillin/streptomycin, 50 µg/mL uridine and 1x GlutaMAX^TM for 20 min. Cells were washed with phenol red-free DMEM (Thermo Fisher Scientific, 21063-029) before incubation in phenol red-free DMEM supplemented with 5% (v/v) FCS, 1x penicillin/streptomycin, 50 µg/mL uridine and 1x sodium pyruvate (Thermo Fisher Scientific), with or without 2 µM rotenone (Sigma) or 4 µM antimycin A (Sigma). Cells were imaged every 15 min using an ArrayScan Vti High Content Analyser (Thermo Scientific, MA, USA), with Hoechst 33342 and MitoSOX^TM fluorescence excited with 365 nm and 549 nm laser lines respectively. Fluorescence measurements were averaged from five wells containing approximately 100 cells per well. Significant differences between treated and untreated control and MCAD ‘knockout’ cells were determined by ANOVA with Tukey’s post-hoc multiple comparison tests.

MitoSOX™ (Thermo Scientific) was used to detect the level of ROS in cells. Cell suspensions were obtained by tryptase digestion and 2′,7′-dichlorofluorescein was used as a probe. After administration, cardiomyocytes were incubated with MitoTracker Green (200 nM, Beyotime, Shanghai, China) and MitoSOX^TM (5 µM, Thermo Scientific, Waltham, MA, USA) at 37 °C in the dark for 30 min, followed by DAPI staining for 10 min and detection of fluorescence intensity using a laser confocal microscope. The excitation and emission wavelengths for MitoTracker Green are 490 nm and 516 nm, respectively. The excitation and emission wavelengths for MitoSOX^TM are 510 nm and 580 nm, respectively. The excitation and emission wavelengths for DAPI staining are 340 nm and 488 nm, respectively.

The production of mitochondrial ROS of the stable cell lines expressing WT, K39R, K39Q, K39E Cytc, and EV were measured using MitoSOX (#M36008, Invitrogen). MitoSOX can selectively enter mitochondria where it fluoresces red (excitation/emission of 510 nm/580 nm) after reaction with superoxide. Cells were seeded at 25,000 cells/well onto 0.1% gelatin coated Costar 96-well plates and cultured overnight in growth media. The next day, cells were washed with 1x PBS and incubated for 30 min at 37 °C with 5 µM MitoSOX in DMEM (#31053-028, Gibco) without phenol red or FBS. After incubation, cells were washed twice with 1x PBS and fluorescence was measured in PBS using a Synergy H1 microplate reader. Mitochondrial ROS production is reported as a percentage of change in fluorescence/µg protein (arbitrary units/µg protein) compared to WT.

Mitochondrial ROS levels were detected by MitoSOX (Invitrogen, #M36008), a fluorescent dye specifically targeting mitochondria superoxide in living cells. HEI-OC1 cells were seeded into a 24-well plate placed with 14 mm cover slip and treated with apoVs or H₂O₂. According to instruction, MitoSOX staining solution were diluted at 1:1000 with preheated Hank's Balanced Salt Solution (HBSS, GIBCO, # C14175500BT). After H₂O₂ treatment, HEI-OC1 cells were washed twice with preheated HBSS and incubated with MitoSOX staining solution for 10 min at 37 °C in the dark. Then HEI-OC1 cells successively underwent fixation, permeabilization, blocking and DAPI staining as above. Samples were observed and imaged with a 60×-magnification lens under fluorescence microscope.

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).

SH-SY5Y cells (6 × 10⁵ cells/well) were added into 12-well plates and incubated at 37 °C and 5 % CO₂ overnight prior to being taken for experimentation. Cells were preincubated with weak base drugs in the absence or presence of DFO (50 µM) for 1 h, washed gently three-times with PBS, and incubated with 2.5 µM of MitoSox (Invitrogen, cat. no. M36008, Carlsbad, USA), which detects superoxide (O₂⁻·) in mitochondria and is a product of the Fenton-like reaction Fe²⁺ + O₂ → O₂⁻· [37 ]. MitoSox and the cells were incubated for 20 min in a 37 °C and 5 % CO₂ incubator. Cells were rinsed three times with warm 1X PBS, fresh PBS was added to the wells, and cells were then dissociated from their culture plates by gentle trituration and then placed in Eppendorf tubes where then the fluorescence was measured using a flow cytometer (Attune NxT, ThermoFisher, Waltham, USA). A minimum of 10,000 cells per condition were collected, and MitoSox fluorescence was analyzed in cells dissociated from the culture plates at an excitation of 510 nm and an emission of 580 nm by flow cytometry (Attune NxT, ThermoFisher, Waltham, USA).