Dcfh da

The SH-SY5Y (1 × 10⁴ cells/well) and HT22 (1 × 10⁴ cells/well) cells were seeded in a 96-well cell culture plate for 24 h, and the primary hippocampal neurons (2 × 10⁵ cells/well) were seeded in a 24-well cell culture plate for 7 days. All cells were exposed to sevoflurane at concentrations of 4 and 8% for 12 h in the presence or absence of NAC. The levels of ROS were evaluated by redox-sensitive dye dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime Biotech, Nanjing, China) as we previously described (Wang et al., 2018 ). Cells were stained with 20 μmol/L DCFH-DA in a dark room for 30 min and the fluorescence density of DCFH-DA was calculated by a fluorescence spectrometer (HTS 7000, Perkin Elmer, Boston, MA, United States).
The levels of ROS in the hippocampi of rat pups were measured using an ROS ELISA detection kit (Jianglai Biotech, Shanghai, China) according to the manufacturer’s instructions. The extracted supernatant of hippocampus tissue samples was added to the ELISA scale, reacted with conjugate for 1 h at 37°C, and then reacted with substrate for 20 min at 25°C followed by stop solution. Then, the fluorescence absorptions were measured by a microplate reader (HTS 7000, Perkin Elmer, Boston, MA, United States) at 450 nm immediately. The concentration of ROS was calculated corresponding to the mean absorbance from the standard curve.

The determination of intracellular reactive oxygen species (ROS) production was followed by the previous report [13 (link)] using 2′,7′-Dichlorofluorescin diacetate (DCFH—DA, Sigma, St. Louis, MO, USA). Briefly, cells were cultured in 96 well plates with DMEM. The culture medium was removed and replaced with 2 µM DCFH-DA in serum-free DMEM in a dark room for 24 h at 37 °C. After H/R protocol, the medium was removed and replaced with a completed DMEM with DCFH-DA and incubated at 37 °C for 1 h. The fluorescence intensity was measured by using an EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

The intracellular generation of ROS was assessed by measuring the oxidation of the probe 20,70-dichlorofluorescin diacetate (DCFH-DA) (Molecular Probes, Lifesciences) [38 (link)]. Briefly, the confluent Caco-2 cells in the 24-well plates were pretreated for 18 h with freeze-dried PJ powder (100 μg/mL). A set of the samples were treated with 10 μM H₂O₂ 2 h before the end of the treatment to induce oxidative stress, and the fluorescence intensity of DCFH-DA (relative fluorescence units) was measured at an excitation and emission wavelength of 485 nm and 520 nm, respectively, using a spectrofluorimeter (Victor3, Perkin-Elmer, Waltham, MA, USA).