2.5. Analysis of Oxidative Stress

1 × 10⁶ ARPE-19 cells per well in 6-well plates were cultured with 2 ml DF-12 medium with 10% FBS. Cells were treated as previously indicated. For ROS assay (Nanjing Jiancheng Bioengineering Institute, E004-1), cells after digestion were prepared into single-cell suspension and divided into negative, positive, and tested groups. The negative group was suspended with 0.01 mol/L PBS, and the positive group was suspended with DCFH-DA together with hydrogen peroxide to induce production of ROS. The tested groups were suspended with DCFH-DA alone. Cells in all groups were suspended into 1 × 10⁶∼2 × 10⁷ cells/ml. After suspension, cells were incubated in 37°C for 30 min. After that, cells were centrifuged under 1000 g for 5 min and washed with PBS once. After removal of the supernatant, cells were resuspended with PBS, and the fluorescence intensity was tested under the fluorescence spectrophotometer.

For SOD assay (Nanjing Jiancheng Bioengineering Institute, A001-3), cells after digestion were prepared into single-cell suspension and washed by PBS once before being smashed in the ultrasonic cell breaker. The mixture was centrifuged at 1000 g at 4°C for 5 min, and supernatant was used for the assay. Reagents and samples were mixed in a 96-well plate as described in the manual. The activity of SOD (U/ml) = 100% × ((OD_(control) − OD_{(blank of control)}) − (OD_(test) − OD_{(blank of test)}))/(OD_(control) − OD_{(blank of control)}).

For MDA assay (Nanjing Jiancheng Bioengineering Institute, A003-1), cells after digestion were prepared into single-cell suspension and washed by PBS once before being smashed in the ultrasonic cell breaker. The mixture was centrifuged at 1000 g at 4°C for 5 min, and the supernatant was used for the assay. Reagents and samples were mixed in a 1.5 ml tube as described in the manual. After mixing, the mixture was heated at 95°C for 40 min and cooled down to room temperature. After centrifugation at 4000 g for 10 min, the supernatant was read at 532 nm. The OD values of the standard and test were corrected by OD of blank. Solutions 1, 2, and 3 were provided by the kit.