Dcfh da

The level of total ROS production in serum was assessed by measuring the oxidation of 2-4-dichlorodihydrofluorescein diacetate (DCFH-DA) (Abcam, Cambridge, MA, U.S.A) by following manufacturer’s manual instructions. In brief, 50 μL of samples were prepared in the 96-well plate. One hundred μL of 10 μM DCFH-DA was added into each well and the plate was incubated for 30 min in the dark. Fluorescence at 488 nm excitation/525 nm emission was analyzed by using DTX-880 multimode microplate reader (Beckman Coulter Inc., Fullerton, CA, U.S.A).

Intracellular ROS were measured by a nonfluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA), which can passively diffuse into cells where it is deacetylated by intracellular esterase to release nonfluorescent 2′,7′-dichlorofluorescein (DCFH). Oxidation of DCFH by ROS yields a highly fluorescent compound dichlorofluorescein (DCF) that is trapped inside the cells^{32 (link)}. The resultant fluorescence intensity of DCF is proportional to the amount of intracellular ROS. In brief, cells were exposed to different energy densities of 830 nm LED irradiation followed by incubation with 10 µM DCFH-DA (Abcam, UK) at 37 °C for 20 min in the dark. The fluorescence intensity of DCF was measured by flow cytometry.

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).

HGC‐27 cells in logarithmic phase were digested and adjusted to cell density of 2 × 10⁵/mL and seeded into 6‐well plates. After the incubation for 2–4 h to allow cell adherence, culture medium was changed into serum‐free medium and cultured for 24 h. Afterwards, cells were treated with PIP in 1640 medium containing 20% FBS for 24 h. DCFH‐DA (Abcam) was used to detect intercellular ROS levels. In brief, after the completion of cell culture, cells were replaced with colourless 1640 medium, followed by staining with DCFH‐DA (50 μM) for 30 min. Inverted fluorescence microscope was used for observation and photographing. ROS was stained as green fluorescence. The number of positive staining cells was counted, and the absorbance was measured by a fluorescence spectrophotometer.

The fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA) was used to detect intracellular ROS production. HEI-OC1 cells, seeded at a density of 5 × 10⁵ and cultured overnight, were treated with 60 μM CDDP alone or co-treated with 1, or 10 μg/ml ATX/ATX-LPN for 24 h, then incubated with 10 μM DCFH-DA (Abcam, MA, USA) in serum-free medium at 37 °C for 30 min in the dark. Quantification of the fluorescence intensity (488 nm excitation/525 nm emission) was performed with a Zeiss confocal laser scanning microscope.