Ros detection kit

The intracellular ROS was measured using a ROS detection kit (Sigma‐Aldrich) was used to detect the intracellular ROS level. The NPCs were treated with compression at 0, 24, 48 hours with or without additional CSA administration. Then, cells were digested and washed by PBS twice, and incubated with 2,7‐dichlorofluorescin diacetate in the dark at 37°C for 30 minutes. Then, ROS production of the cells was measured by a flow cytometry (BD LSR II, Becton Dickinson), following the manufacturer's instructions.

β-Carotene was purchased from Sigma-Aldrich (Cat.no.7235-40-7). CD73 (ab202122, 1:200 dilution), CD90 (ab23894, 1:500 dilution), CD105 (ab2529, 1:200), P16 (ab220800, 1:1000), and P15 (ab53034, 1:500 dilution) were purchased from Abcam (United Kingdom). DMEM/F-12, fetal bovine serum (FBS), and non-essential amino acids (NEAA) were purchased from Thermo Fisher. Cell cycle and apoptosis detection kit and cell aging β-Galactosidase staining kit were purchased from Biyuntian (Shanghai, China). DNA extraction kit was purchased from Tiangen; Osteogenic induction differentiation medium and adipogenic induction differentiation kit were purchased from Thermo Fisher. ROS detection kit was purchased from Sigma-Aldrich. CCK-8 kit was purchased from Dojindo Laboratories (Kumamoto, Japan).

HaCaT cells were cultured for 24 h in the presence (or absence) of 400 µg/mL UD and 0.001% and 0.005% ZP. Following incubation, HaCaT sample medium was replaced with a ROS detection kit (Sigma-Aldrich, Darmstadt, Germany), and samples were irradiated for 25 min with UVA (6.9 J/cm²: Luzchem irradiator LZC-420). Non-irradiated and non-treated controls were maintained in the dark at 37 °C during this period. Reactive ROS concentrations in the test samples were measured 2 h later by spectrophotometry, as described above. Cell viability analysis by MTT was also performed to assess UD and UD + UVA-induced cell mortality, normalizing ROS levels to the quantity of living cells in each specific condition.
For the analysis of results, the blank control was subtracted from the sample data. Then, relative ROS levels with respect to the cell survival average were calculated from each condition and normalized with respect to control + UD + UVA. These values were represented as mean ± SEM, analyzed statistically (Students t-test) comparing UD + UVA-treated samples versus UD + UVA control samples. Statistical significance was set at p < 0.05 (95% confidence).

Oral cancer cells were treated with 1 mM SSZ for 8 h. Then the cellular ROS and lipid ROS were detected using a ROS detection kit (Sigma-Aldrich) according to the instructions of the manufacturer. Cellular glutathione (GSH) levels were measured using a GSH colorimetric detection kit (Sigma-Aldrich). Cellular lipid peroxidation was also evaluated by determining the malondialdehyde (MDA) concentration using a lipid peroxidation detection kit (Sigma-Aldrich).

SKOV3 and HO8910 cells (1.0 × 10⁵ cells/mL) were seeded in 35 mm culture dishes and supplied with complete medium. After attachment, the cells were exposed to SMFs for 12 or 24 hours. ROS detection kit (Sigma, USA) containing 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect cellular ROS. Cultured cells were incubated with 10 μM DCFH-DA at 37°C for 30 minutes before their ROS levels were evaluated using flow cytometry.