Dcfh da ros assay kit

The mouse hepatocyte cell line AML12 was treated with CoCl₂ (Sigma-Aldrich, St. Louis, USA) to establish the cell hypoxia model as described previously [29 (link)]. AML12 cells were cultured in DMEM/F12 (Hyclone, Logan, USA) containing 10% fetal bovine serum (FBS), ITS liquid media supplement (Sigma-Aldrich, St. Louis, USA), and 40 ng/mL dexamethasone (Solarbio, Beijing, China) at 37°C with 5% CO₂. The cells divided into four treatment groups. These were the control, ORY (240 μg/mL), CoCl₂ (300 μM), and CoCl₂+ORY (240 μg/mL ORY was added at the same time with CoCl₂ (300 μM)) groups [30 (link)]. Levels of ROS in AML12 were determined by DCFH-DA ROS assay kit (Beyotime, Shanghai, China) according to the manufacturer's instruction. The mouse hepatocyte cell line AML12 was treated with tunicamycin (TM) (Solarbio, Beijing, China) as described previously [31 (link)]. These were the control, ORY (240 μg/mL), TM (10 μg/mL), and TM+ORY. The cells were pretreated with γ-oryzanol for 12 h, followed by treatment with 10 μg/mL tunicamycin for an additional 24 h.

KGN cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) after H₂O₂ and/or treatment with 20 µmol/L BAI. Briefly, KGN (100 µl, 1 × 10⁴ cells) cells were inoculated in a 96-well plate and cultured at 37 ℃ in a conditioned incubator containing 5% CO₂. After treatment for 24 h, 10 µL of CCK-8 was added to every well, and the culture plate was incubated in a multiscan MK3 incubator (Thermo Fisher Scientific, Waltham, MA, USA) for 4 h. Absorbance was measured at 450 nm using a microplate reader. KGN cell apoptosis was analyzed using an Annexin V/PI apoptosis kit (BD, Franklin Lakes, NJ, USA). In brief, KGN was washed twice with cold PBS and then resuspended cells in 1X Binding Buffer at a concentration of 1 × 10⁶ cells/ml. Then, 100 µl of the solution (1 × 10⁵ cells) was transferred to a 5 ml culture tube and added 5 µl of FITC Annexin V and 5 µl PI. KGN was gently vortexed and incubate for 15 min at 25 °C in the dark. Finally, 400 µl of 1X Binding Buffer was added to each tube and a FACSCalibur flow cytometer (BD). Reactive oxygen species (ROS) level in KGN cells was analyzed using a DCFH-DA ROS Assay Kit (Beyotime, Shanghai, China) and FACSCalibur flow cytometer (BD). All reactions were performed in triplicate.

Oxidative stress was evaluated with reactive oxygen species (ROS) and malondialdehyde (MDA) generation and antioxidant enzymes (total antioxidant capacity, T-AOC; superoxide Dismutase, SOD; total Glutathione S-transferase, TGST). The A549 cells in the inner chamber of each group were harvested and lysed by ultrasonication in the presence of a protease inhibitor. The supernatant was collected for analysis after centrifugation at 14,000 × g for 5 min. The levels of MDA, T-AOC, SOD and TGST in the supernatant were measured using appropriate kits (Beyotime, China) following the manufacturer's instructions.
ROS was measured using the DCFH-DA ROS assay kit (Beyotime, China). Cells were labeled with a 10 µM probe (2,7-dichlorofluorescin-diacetate, DCFH-DA) at 37° C for 30 min in the dark. Then, the cells were washed with PBS and analyzed on an enzyme-labeled instrument by a fluorescence spectrophotometer with an excitation wavelength (488 nm) and an emission wavelength (530 nm).