H2dcfda

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).

Yeast cells were grown till OD₆₀₀ ~ 0.8, cells were washed with PBS, followed by incubation in PBS containing 100 µM H₂DCFDA dye (Sigma # D6883) and 5 µM MitoSOX Red (Invitrogen #M36008) for 30 min in the dark at 37 °C followed by washing with PBS. Slides were mounted and observed under Leica SP8 confocal microscope using a 63× objective lens with the FITC (Em λ₅₂₉) filter to measure H₂DCFDA fluorescence and TRITC (Em λ₅₈₀) filters to measure MitoSOX Red fluorescence. The images were quantitated using NIH ImageJ software (1.52p).

Intracellular ROS production was assessed by 2′,7′ dichlorodihydrofluorescein diacetate (H₂DCFDA) (Molecular Probes, Eugene, OR, USA) staining, as previously reported [54 (link)]. HCT116 cells (7 × 10³ cells/well) were seeded in 96-well plates and incubated with TBT-F (400 nM) for 3 or 24 h. After treatment, cells were washed with PBS, containing 5 mM glucose and stained with H₂DCFDA (20 µM final concentration) at 37 °C for 30 min in the dark. Cells were then washed with PBS and H₂DCFDA-positive cells were analyzed by fluorescence microscopy using an appropriate fluorescein isothiocyanate (FITC) filter (Leica DMR, Microsystems S.r.l, Wetzlar, Germany). All images were acquired by a computer imaging system (Leica DC300F camera) and three different visual fields were examined for each condition. The fluorescence intensity was estimated by a Varian CARY Eclipse Fluorescence Spectrophotometer (Varian Medical Systems Italia SpA, Milan, Italy). Data reported represent the means of three independent experiments. ROS generation was measured in a fluorescent plate reader (excitation at 488 nm and emission at 520 nm). Cells incubated with 10 µM H₂O₂ were used as a positive control (n = 9 per condition).

Trichogin GA IV-derived peptides at the concentration of 50 μM in water were applied as 10-μl droplets on the abaxial surface of detached Micro-Tom and A. thaliana leaves. Five droplets were applied on each leaf, and three leaves per treatment were analyzed. Incubations were performed in Petri dishes for 24 and 48 h as previously described. ROS production by leaves was visualized by staining with the cell-permeable probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Sigma-Aldrich, United States), as reported in Luti et al. (2020) (link). For staining, leaves were soaked for 1 h in a 10 μM H₂DCFDA solution in 20 mM phosphate buffer, pH 6.8. After H₂DCFDA incubation, leaves were washed twice with phosphate buffer and mounted on glass slides for microscopy analysis, which was performed under a confocal Leica TCS SP5 scanning microscope (Leica, Germany; λex 460 and λem 512 nm).

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).

Guinea pig aortic endothelial cells seeded on glass coverslips were loaded with the ROS‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen, 2.5 μmol/l) – dissolved in 0.1% DMSO and Pluronic acid F‐127 (0.01% w/v) – added to cell culture media for 15 min. at 37°C 48. The cells were fixed in 2% paraformaldehyde for 10 min. at room temperature and the H₂DCFDA fluorescence analysed using a Leica TCS SP5 confocal laser scanning microscope with 488‐nm excitation wavelength and ×63 oil immersion objective. Intracellular ROS levels were also monitored by flow cytometry: single‐cell suspensions were incubated with H₂DCFDA (1 μmol/l) for 15 min. at 37°C and then analysed using a FACSCanto flow cytometer (Becton‐Dickinson).

BEAS-2B cells were incubated on a cover glass in the presence or absence of 50 μg/mL PM₁₀ in PSS containing 10 μg/mL ROS fluorescence probe 5-(and-6)-choloromethyl-2′,7′-dicholorodihydrofluorescin diacetate (CM-H2DCFDA, Invitrogen) for 5 min and washed for 5 min in PBS. H2DCFDA fluorescence was measured using a confocal laser-scanning microscope (Leica, Buffalo, NY) by excitation at 488 nm and measuring the emitted light at 525 nm. H2DCFDA fluorescence was collected for six different regions in each image, and the signal was normalized to that at the beginning of the experiment.