Copper-induced oxidation of LDL

Prior to LDL oxidation, ethylene diamine tetraacetic acid (EDTA) was removed from LDL solutions via dialysis at 4 °C for 36 h. For oxidation, native LDL solutions were mixed with cupric sulphate (99.0% purity; Jinshanting New Chemical Reagent Factory, Shanghai, China) at a final copper concentration of 5 µM in phosphate-buffered saline (PBS) at 37 °C for indicated periods of time. The oxidation was terminated by 5 µM EDTA. After removing EDTA and cupric sulphate via centrifugation at 10,000×g at 4 °C for ~ 15 min by using the Amicon Ultra-0.5 ml, 100 kDa Centrifugal Filter Unit (Merck Millipore), the oxLDL samples were subjected to the following experiments. The concentration of LDL/oxLDL was determined by bicinchoninic acid (BCA) assay and adjusted with sterile PBS.

For the dynamic AFM detection of LDL oxidation, the functionalized mica sheet pre-immobilized with native LDL particles was put on the sample stage of atomic force microscope; after adding the cupric sulphate solution at a final copper concentration of 5 µM in PBS, the LDL/oxLDL particles were imaged by AFM immediately and the imaging lasted for around 16 h.