Sod detection kit

Production of intracellular ROS was assessed using a fluorescent probe 2’, 7’-dichlorofluorescein diacetates (DCFH-DA) (Beyotime, Wuhan, China). Briefly, cells were treated with A1254 (0, 2.5, 5, 10, and 20 μmol/L) alone or A1254 (10 μmol/L) + NAC (2 mmol/L) for 24h or 48h, with 0.1% DMSO (v/v) treatment as negative control. Then, MC3T3-E1 cells were incubated with 10 μmol/L of DCFH-DA for 30min. The fluorescence image was obtained using a microplate reader (Olympus, Tokyo, Japan) at an excitation 485 nm and emission 530 nm. The fluorescence intensity was quantitated using the Ipwin32 software (30 (link)).
SOD activity and MDA levels after A1254 exposure were determined using a SOD detection kit (S0101M, Beyotime, Shanghai, China) and MDA assay kit (S0131M, Beyotime, Shanghai, China), respectively. The SOD activity was calculated through colorimetric analysis of nitroblue tetrazoliumusing using a plate reader (Multiscan Mk3, Thermofisher, Massachusetts, USA) at 450 nm, and MDA levels were measured based on thiobarbituric acid chromogenic reaction using the Multiscan Mk3 plate reader at 535 nm.

The SOD detection process was carried out strictly in line with the instructions of SOD detection kit (S0101S; Beyotime, China). Briefly, HaCaT cells were planted onto a 12-well plate (2 × 10⁵ cells / mL) and the cell lysates were prepared with PBS. Skin tissues were placed in cold saline and homogenized through a homogenizer machine, followed by centrifugation at 3000 g lasting 15 min to obtain the supernatant. SOD levels were detected in HaCaT cells and 10% skin tissue homogenate.