Mda assay kit

Mature C57BL/6 mice with 8-week-old was supplied by Guangdong Medical Laboratory Animal Center (license number: SYXK(Yue)2008–0002) and maintained in a standard pathogen-free environment with a controlled diet and a controlled temperature (23–25)°C. Laboratory animal experiments performed were approved by the Hangzhou Red Cross Hospital Animal Care and Use Committee.
Qingfei Xieding was available from Hangzhou Red Cross Hospital. The mixture of Ephedra sinica (9 g), apricot kernel (9 g), gesso (18 g), lobed kudzuvine root (9 g), Scutellaria baicalensis (9 g), Bombyx batryticatus (15 g), and Houttuynia cordate (3 g) was decocted in 1.5 L water till condensing to 300 ml. After removing the dregs, extracts were collected and stored at 4°C.
Bleomycin sulfate was purchased from Sigma (St. Louis, MO, USA). HYP, NO, T-AOC, and MDA assay kits were obtained from Beyotime (Jiangsu, China). Antibodies used in this study were purchased from Abcam (Cambridge, British). RNeasy mini kit and qRT-PCR assay kit were obtained from Qiagen (Shanghai, China) and Takara (Dalian, China), respectively.

Malondialdehyde (MDA) assay kits (Beyotime, S0131M) were used to assess the level of lipid peroxidation according to the manufacturer's guidelines. Proteins were obtained from 30 mg of tumor tissues using a radioimmunoprecipitation assay (RIPA) protein extraction reagent (Beyotime, P0013B). The protein concentration was determined using a BCA protein assay kit. Then, 100 mL of the protein sample or standard protein was incubated with 200 mL of working fluid for MDA detection for 15 min at 100 °C. The samples were then cooled to room temperature and centrifuged at 1000 g for 10 min at room temperature to obtain the supernatant. Finally, 200 µL of the supernatant was added to the 96-well plate, and the absorbance was measured at 532 nm with a microplate reader. The MDA level was represented as the ratio of absorbance value to the control group.

Lipid peroxidation levels were detected by malondialdehyde (MDA) assay kits (Beyotime Biotechnology, 532 nm), 4-HNE assay kits (ab238538, Abcam Inc., Cambridge, MA, USA, 450 nm), and a fluorescent lipid peroxidation probe (C11 BODIPY 581/591, Shanghai Mao Kang Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The absorbance was measured by a microplate reader (Bio-Tek Instruments), and the fluorescence was determined using a fluorescence microscope (Olympus, Tokyo, Japan).