Reactive oxygen species fluorometric assay kit

About 25 mg fresh tumor tissue was homogenized with lysis buffer [2% SDS (Sigma), and 1× protease inhibitor (Roche) in PBS] to obtain tumor tissue lysate. The level of malondialdehyde (MDA) in tumor tissue lysates was evaluated according to the instructions of Lipid Peroxidation Assay Kit (Cayman, Ann Arbor, USA). Then, the absorbance at 532 nm was measured using spectrophotometer. Intracellular ROS of tumor tissue was assessed according to the instructions of Reactive Oxygen Species (ROS) Fluorometric Assay Kit (Elabscience, Wuhan, China). In brief, tumor tissues were digested into single cells and incubated with DCFH-DA for 1 h at 37 °C in the dark, and washed three times with PBS subsequently. Then, the fluorescence intensities reflecting the intracellular ROS were detected by spectrophotometer at 525 nm absorbance. For lipid peroxidation study, the cells were incubated with 10 μM sensor probe BODIPY(581⁄591)-C11 (Thermo Fisher Scientific) for 1 h at 37 °C. Then, Excess BODIPY-C11 probe was removed by washing the cells twice with PBS. Labeled cells were trypsinized, and then re-suspend in PBS containing 5% BSA. The fluorescence intensity of each sample was determined by flow cytometry (BD FACSVerseTM, BD, USA). The shifted fluorescence of emission peak from 590 to 510 nm in each sample was analyzed and used to calculate the relative lipids.

The cellular ROS levels were measured using the Reactive Oxygen Species (ROS) Fluorometric Assay Kit (Elabscience, Wuhan, China) according to the product instructions. The values of ROS were detected by a fluorescence microplate (Thermo). The wavelength of excitation and emission was 500 nm and 525 nm, respectively.