Ros detection kit

Podocytes were cultured in a 24-well plate. Upon reaching 80% confluence, the podocytes were treated with different media for 24 h. ROS detection was performed using an ROS detection kit (Beyotime, Haimen, China) according to the manufacturer's protocol. The podocytes were incubated with 10 μM DCFH-DA for 20 min at 37°C. The fluorescence intensity was observed and recorded by a fluorescence microscope.

The ROS production, malondialdehyde (MDA) level, SOD and catalase (CAT) activity, and Fe²⁺ in the cells under different treatments were measured using the corresponding kits. The cells were incubated with 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA) probe in the dark for 30 min in line with the instructions of the ROS detection kit (Beyotime, Shanghai, China). Then the fluorescence intensity was observed under the fluorescent microplate. The laser wavelength was 485 nm, and the emission wavelength was 525 nm; ROS level (%) = fluorescence value of intervention group/control group × 100%. CAT activity was detected using visible spectrophotometry and CAT detection kit (A007‐1‐1; Jiancheng Bioengineering Institute, Nanjing, China). MDA level was determined using the thiobarbituric acid method and MDA detection kit (A003‐1‐2; Jiancheng Bioengineering Institute). SOD activity was detected using the hydroxylamine method and total SOD detection kit (A001‐1‐2; Jiancheng Bioengineering Institute). The absorbance value at 593 nm was measured to calculate the iron ion level in line with the instruction of Fe²⁺ iron ion kit (MAK025; Sigma‐Aldrich).

The relative levels of intracellular ROS were measured using a commercial ROS detection kit (Beyotime, China). Briefly, IPEC-J2 cells were cultured in 6-well plates for 16 h before treatment, pretreated with RSV (0 μM, 25 μM, and 50 μM) for 4 hours, and then exposed with H₂O₂ (500 μM) for 4 h to induce ROS production. Cells were washed twice with PBS and then incubated with 5 μM 2′,7′-dichlorofluorescein diacetate (DCFHDA) for 30 min. Fluorescence intensity was measured using a fluorescence microplate reader (Tecan, Sunrise) at excitation/emission wavelengths of 525/610 nm.

The cellular ROS accumulation was measured with an ROS detection kit (Beyotime Biotechnology, Shanghai, China), according to the manufacturer’s instructions. In brief, the cells were seeded in 6-cm culture dishes, treated with 2 μM afatinib for 24 h, and were then washed twice with 1× PBS. Next, 2 × 10⁵ cells were collected and incubated with 10 μM DCFH-DA for 20 min. The cell pellet was suspended with 500 μL 1× PBS and cytometry analysis was performed to detect fluorescent signal intensity with excitation at 488 nm and emission at 525 nm. Flow cytometry data were analyzed using Cell Quest software.

To determine changes in intracellular ROS, BMSCs were seeded in 6-well plates at a density of 2.5 × 10⁴ cells/well, with or without different concentrations of D-gal or NAD⁺ or knocked down Sirt1 by si-RNA, and then incubated at 37 °C for 48 h. According to the instructions for the ROS detection kit (Beyotime, Shanghai, China), after removing culture media, the cells were washed once with PBS, then added to a medium containing 10 μmol/L DCFH-DA (Beyotime, Shanghai, China) that was added to plates and then incubated for 30 min. The media were then removed, followed by washing three times with PBS. After trypsinization, the digestion was terminated with PBS containing 0.2% FBS and intracellular ROS levels were detected by flow cytometry (BD, New York, NY, USA). These results were analyzed using Flowjo-V10.