A106 1

MDA (A003-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and LPO (A106-1, Nanjing Jiancheng Bioengineering Institute) in vascular tissues or cells were measured using commercial kits, according to the manufacturer’s instructions. Briefly, cells (1 × 10⁶) were collected in 200 μL of lysis buffer and homogenized on ice. Subsequently, an MDA test solution (1000 μL) was added to each experimental sample or vial containing the standard sample to form an MDA-TBA adduct, followed by incubation for 40 min at 95 °C. For LPO analysis, 800 μL of the LPO test solution was added to each experimental sample or vial containing the standard sample and then incubated for 60 min at 45 °C. The mixture was cooled to 25 °C in an ice bath and centrifuged at 4000 rpm for 10 min. The insoluble material was removed, and 250 μL of each reaction mixture was pipetted into 96-well plates for colorimetric assays to measure the absorbance of MDA and LPO at 530 nm and 586 nm, respectively, with an Infinite M200 Pro NanoQuant (TECAN).

RAW264.7 macrophages were seeded in 96-well plates (3 × 10³ cells/well) and treated with several experimental treatments 24 h after cell adhesion. After stimulation, a lipid peroxide test kit (A106-1, Jiancheng, Nanjing, China) was used to evaluate the amount of lipid peroxide in each group. A microplate reader (Multiskan FC, Waltham, MA, USA) was used to measure the absorbance at 586 nm.
RAW264.7 macrophages were seeded in 6-well plates (1 × 10⁶ cells/well) and treated with several experimental treatments 24 h after cell adhesion. Lipid peroxidation levels were detected using a C11BODIPY probe (D3861, Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated with 2 μM C11BODIPY for 30 min at 37 °C, then washed three times with PBS. Finally, cells were imaged using a fluorescence microscope (THUNDER DMi8, LEICA, Munich, Germany). The mean fluorescence intensity of C11BODIPY Green, which measures the amount of lipid ROS in the cell, was calculated using ImageJ software (NIH, Bethesda, MD, USA).