Dcfh da

2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA; Funakoshi, Tokyo, Japan) stock solution of 1 mg was prepared in 100 µL ethanol and stored at -20 °C. Yeast cells (10⁶ cells/mL) were inoculated in 10 mL minimal synthetic medium containing 15% (m/V) glucose with or without amino acids and incubated for 2–3 days at 30 °C with liquid paraffin overlaid on the culture. After fermentation, 10⁷ cells/mL yeast cells were recovered and incubated with 13 µL DCFH-DA, 100 µL phosphate-buffered saline (PBS), pH=7.4, and 887 µL sterile water at 30 °C for 20 min in a shaker (H500-H; Benchmark Scientific Inc., Sayreville, NJ, USA) at 200 rpm. After incubation, the cells were washed three times with sterile water, centrifuged (KUBOTA 5200; Kubota, Co. Ltd.) at 13 000×g for 1 min and 100 cells were sampled under fermentation stress, treated with DCFH-DA and the ROS content was analyzed by fluorescence microscopy (Olympus BX53; Olympus, Tokyo, Japan).

Intracellular ROS were measured using 2′,7′‐dichlorofluorescein‐diacetate (DCFH‐DA, Sigma–Aldrich) as described.²⁶ BV2 cells were treated with or without BGN‐shRNA (sh‐NC for control) prior to LPS (1 µg/ml, 8 h) and ATP (5 mM, 2 h) exposure. Then, DCFH‐DA (10 µM) was added and incubated for 0.5 h at 37°C prior to washing three times using serum‐free medium. An Olympus fluorescence microscope (Tokyo, Japan) was used to observe the staining of DCFH‐DA (485 nm excitation, 535 nm emission), and flow cytometry was applied to analyse the ROS production in three random samples of each group.

The malondialdehyde (MDA), NADPH oxidase and the total superoxide dismutase (SOD) were detected as indicated by manufacturer (Biotein, Shanghai, China). ROS was detected by dichlorofluorescein diacetate assay (DCFH‐DA, Biotein, Shanghai, China). The cells were incubated with DCFH‐DA (10 μmol/L) for 60 minutes at 37°C, and immunofluorescence was detected using a fluorescence microplate reader (excitation wavelength/emission wavelength: 485/525 nm) or by light microscopy (BX51TRF; Olympus Corporation, Tokyo, Japan).

Levels of intracellular ROS were examined by the use of the fluorescent probe 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) (Sigma-Aldrich, Poznan, Poland). The samples of each group were loaded with DCFH-DA (10mM) at 37°C in the dark and then rinsed three times before observation of intracellular ROS fluorescence under an Olympus BX51 fluorescence microscope (Tokyo, Japan). The fluorescence intensity reflected the generation of intracellular ROS among different groups.

AC16 cells were seeded into 6-well plates and treated with 35 mmol/L glucose and PQQ for 24 h. To measure intracellular reactive oxygen species (ROS) concentrations, the cells were incubated with 10 μmol/L 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA; Abcam, Cambridge, MA, USA) for 45 min at 37 ℃. The cells were then washed three times with serum-free medium, and DCFH-DA fluorescence was observed using a fluorescence microscope (Olympus Corporation; magnification 400×). Fluorescence intensities were quantified using Image-Pro Plus (version 5.0; Media Cybernetics, Inc., Rockville, MD, USA).