2 7 dichlorofluorescin diacetate dcfh da probe

ROS production was measured using the 2′-7′-dichlorofluorescin diacetate (DCFH-DA) probe (Sigma-Aldrich, Madrid, Spain). Cells were seeded in 96-well culture plates. Then, the cells were incubated with 100 μM DCFH-DA in culture medium without phenol red for 30 min at 37°C and then rinsed with PBS and filled with Krebs-Ringer bicarbonate buffer. ROS levels were measured in a multiwell plate reader spectrofluorometer (BioTek Instruments Inc., Winooski, VT, USA) for 45 minutes each 5 minutes at 485 nm to excitation and 530 nm to emission [18 (link)].

ROS, GSH and MDA levels, SOD activity were all determined using commercial detection kits (Jiancheng Biotech, Nanjing, China) following the manufacturer's manuals. Briefly, GSH activity was measured by the reaction of 5.50-dithiobis-2-nitrobenzoic acid (DTNB) with GSH, the product of which can be read at 412 nm using a spectrophotometer. Binding of MDA with thiobarbituric acid results in a chromogenic complex that can be detected at 532 nm. SOD activity can be measured spectrophotometrically based on inhibition reaction of NADPH-phenazinemethosulphate-nitroblue tetrazolium formazan, as enzyme reaction can be detected by recording the absorption at 560 nm. Intracellular ROS level was measured using the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe (Sigma) in a detection kit. The ROS level was measured as the absorbance at 525 nm recorded with a spectrometer.