Dcf da

Intracellular ROS levels were assessed using the ROS-sensitive fluorescent dye DCFDA (Sigma-Aldrich). The cells were incubated with 2.5 μM DCFDA for 15 min. For the detection of mitochondrial ROS production, we used the mitochondrial-sensitive dye MitoSOX Red (Molecular Probes, Eugene, OR, USA). Hippocampal NSCs were incubated with 3 μM MitoSOX Red for 10 min. Fluorescence was captured using a 40× objective lens on a Carl Zeiss LSM 700 Meta confocal microscope (485-nm excitation and 535-nm emission for DCFDA; 510-nm excitation and 580-nm emission for MitoSOX Red). DCFDA and MitoSOX Red fluorescences were quantified from cells of interest using the measurement functions on the Carl Zeiss confocal software.

For quantitative evaluation of ROS generation, cells were stained with 10 μM 2’,7’-dichlorofluorescein diacetate (DCF-DA, Thermo Fisher Scientific) and then the intensity of DCF fluorescence reflecting ROS production was analyzed as described previously [18 (link)]. In addition, DCF-DA fluorescence images were captured under a fluorescent microscope (Carl Zeiss, Germany).

Dynamic analysis of cellular redox was carried out using four different live cell imaging dyes, that detect specific molecular species: cytoplasmic superoxide is analysed using DHE (Life Technologies, D11347); mitochondrial superoxide is detected with mitoSOX™ Red (Life Technologies, M36008); global cellular hydrogen peroxide (H₂O₂) and peroxyl radicals (HO₂) are measured using DCFDA (Sigma, D6883) while unbound reduced glutathione (GSH) is assayed with MCB (Life Technologies, M-1381MP). Cells were cells co-transfected with an appropriate fluorophore that would not interfere with the fluorescence of the dye. All dyes were administered to the recording medium (RM) onto cells plated on coverslips, within Attafluor® metal cell chambers (Molecular Probes™, Thermo fisher, A-7816). RM composition is: 5.6 mMKCl (Sigma, P9333), 10 mM D-(+)-Glucose (Sigma, G7528), 10 mM HEPES (Sigma, H4304), 4.2 mM NaHCO₃ (Sigma, 56297), 138 mM NaCl (Sigma, S5886), 2.6 mM CaCl₂ (Sigma, C7902), 1.2 mM NaH₂PO₄ (BDH,1024940), 1.2 mM MgCl₂ (BDH, 10149), pH 7.4.
DHE (10 μM), mitoSOX (10 μM), and DCFDA (15 μM) were diluted in RM and imaged onto a Zeiss LSM510 confocal microscope. MCB was used at final concentration of 2.5 μM in RM. Brightness controlling settings were maintained consistently within the experiment for all techniques.

ROS production was quantified by fluorescence microscopy (ZEISS, Oberkochen, Germany) using a 2′,7′-dichlorofluorescein diacetate probe (DCF-DA). Mouse VSMCs were incubated with 10 μM of DCF-DA under dark conditions for 30 min at 37°C, and rinsed with phosphate‐buffered saline (PBS). ROS production was measured using an ELISA plate reader (Molecular Devices) at 488 nm excitation and 522 nm emission wavelengths.

Intracellular H₂O₂ production was measured using 5- and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA; Molecular Probes, Eugene, OR, USA) as previously described^{48 (link)}. Briefly, the cells were grown in serum-starved McCoy’s 5A medium supplemented with 1% FBS for 24 h. The cells were then switched to serum-free DMEM without phenol red and exposed to LCA for 30 min. The cells were treated with 10 mM metformin or 1 mM NAC 1 h prior to the LCA treatment to assess their effects on ROS production activated by LCA. The cells were incubated with 5 ng/ml DCFDA for 15 min and immediately observed using an LSM 510 laser-scanning confocal microscope (Carl Zeiss, Germany). The DCF fluorescence was excited at 488 nm using an argon laser, and the emission evoked was filtered with a 515 nm longpass filter.
All obtained fluorescence images taken with the LSM 510 confocal microscope were analyzed using the LSM 5 Image Browser software.

Cells were exposed to 25-μM purpurin or 200-mM DMSO immediately after treatment with 1-mM H₂O₂. For reactive oxygen species (ROS) formation, 20-μM 2′,7′-dichlorofluorescein diacetate (DCF-DA, Invitrogen Molecular Probes, Eugene, OR, USA) was added to HT22 cells at 10 min after H₂O₂ treatment to induce the formation of DCF, which has strong fluorescence. Cells were harvested 30 min after DCF-DA treatment. DNA fragmentation was validated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as described in previous studies [22 , 23 (link)]. Briefly, cells were harvested at 3 h after H₂O₂ treatment, and DNA fragmentation was visualized using a TUNEL staining kit (Sigma). Microphotographs from DCF-DA and TUNEL staining were taken using a confocal fluorescence microscope (LSM 510 META NLO; Zeiss GmbH, Jena, Germany), and the fluorescence intensity was measured using a Fluoroskan ELISA plate reader (Labsystems Multiskan MCC/340). Cell death was assessed using a WST-1 assay at 5 h after H₂O₂ treatment, and formazan fluorescence was measured using a Fluoroskan ELISA plate reader.