Dcfh da

The level of intracellular ROS in HT22 cells incubated with glutamate alone or treated with corresponding compounds was evaluated according to the instructions of the redox‐sensitive dye DCFH‐DA (Beyotime Biotech). Cells were washed twice with PBS, stained with DCFH‐DA (20 μmol/L) for 30 min in the dark, and then analyzed by flow cytometry (CytoFLEX S, Beckman Coulter Inc.). Cells were visualized under a fluorescence microscope (Olympus IX71).

MEF cell lines were seeded in 96-well cell culture plates (SPL Life Sciences Co., Pocheon, Korea) at 1.5 × 10⁴ cells/well and pretreated with BG (0, 1, 10, 50, 100, 250 µg/mL) for 23 h, then subsequently stimulated with 2 mM H₂O₂ for 1 h to induce ROS production. Then 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich Co., Saint Louis, MO, USA) was added to the cells. After 1 h incubation at 37 °C in the dark, the florescence of DCFH-DA was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm by using a fluorescence multimode detector (DTX800, Beckman Coulter, Inc., Brea, CA, USA).
For ROS measurement in vivo, zebrafish embryos at 8 hpf were isolated in 24-well plates at 10 embryos/well with egg water supplemented with 0.1% methylene blue, treated with BG (0, 1, 10, 50, 100, 250 µg/mL) for 1 h, and 5 mM H₂O₂ was added for an additional 24 h to induce ROS. After incubation, the embryos were washed with egg water and grown to 2-day post fertilization (dpf). At 2 dpf, the eggs were treated with egg water containing DCFH-DA (20 μg/mL), incubated for 1 h at 28 °C in the dark, and then washed with egg water. Images of stained embryos were observed using a digital microscope (Dino-Lite Digital Microscope, ANMO Electronics Co., New Taipei City, Taiwan).

The probes from 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Molecular Probes, Eugene, OR, USA) will be used to evaluate the intracellular H₂O₂ contents [17 (link)]. After incubation of orbital fibroblasts with 20 μM DCFH-DA at 37°C for 20 min, cells were trypsinized and then resuspended in 0.5 mL of PBS buffer (pH 7.4) and analyzed with a flow cytometer (Model EPICS XL-MCL, Beckman-Coulter, Miami, FL, USA). The excitation wavelength is set at 488 nm and the intensity of emitted fluorescence of a total of 10,000 cells at 525 nm is recorded on channel FL1 for the DCFH-DA probe. Data were analyzed by the EXPO32™ software (Beckman-Coulter, Miami, FL, USA). The intracellular H₂O₂ contents in the treated cells were presented as relative values compared with that of the cells without H₂O₂ or CSE treatment.

DCFH-DA (20,70-dichlorodihydrofluorescein-diacetate) stock solution (Calbiochem, Germany) was prepared using DMSO and kept in the dark at 20 °C. On culture dishes (60-mm), BMDMs were primed with 50 µM of ECH or CL-ECH (5, 10, 25, 50 µM) for 6 h and then added with 100 ng/mL of LPS (lipopolysaccharides from Salmonella enterica serotype Minnesota; Sigma-Aldrich, St. Louis, MO, USA) when the confluence reached 90%. After 24-hour LPS stimulation, the cells were treated with 10 M of DCFH-DA for 30 min. The green fluorescence of DCFH-DA was recorded at 515 nm with flow cytometry (Beckman Coulter).

ROS production was analysed comprehensively by individually measuring total ROS using the 2,7-dichlorofluorescein diacetate (DCFH-DA, Life Technologies) probe, mitochondrial superoxide production using the MitoSox Red (Life Technologies) probe and superoxide using the dihydroethidium probe (DHE, Life Technologies). DCFH-DA working solution was added directly to the medium to reach 10 μM and then incubated at 37 °C for 15 min in the dark; 50 nM MitoSox Red and 20 μM DHE were used to individually stain cells for 30 min at 37 °C in the dark. All stained cells were washed twice with PBS, resuspended in PBS and kept on ice for immediate detection by flow cytometry (FC500, Beckman Coulter) at the optimal excitation/emission wavelengths (DCFH-DA at 485/535 nm; MitoSox Red at 510/580 nm; DHE at 480/576 nm). Data were consistently analysed using FlowJo software (Ashland, OR, USA).