Oxidized Low-density Lipoprotein (OxLDL) and DiI-OxLDL

Human low-density lipoprotein (LDL) is one of the key lipid-protein complexes in blood and is a crucial component of metabolism responsible for the transport of lipids throughout the body. LDL is composed of cholesterol, cholesterol esters, triglycerides, phospholipids, and a single Apo B-100 protein. LDLs deliver fatty acids and cholesterol to peripheral and liver cells through receptor-mediated endocytosis. High levels of LDL cholesterol are considered a risk factor for atherosclerosis and cardiovascular disease, while high levels of high-density lipoproteins (HDLs) may reduce the risk.

Oxidation of LDL is a natural process within the body caused by free radicals, however excess oxidized LDL can be harmful. Alterations in lipid metabolism such as oxidation of LDL can lead to inflammation in arteries and atherosclerosis. Atherosclerosis is a type of arteriosclerosis in which arterial wall thickening is due to the buildup of fatty plaques, composed of cholesterol and other lipids, that can inhibit and block blood flow in vessels. Endothelial cells and macrophages are recruited to remove oxidized LDL (OxLDL), which initiates inflammatory and immunogenic responses. Unlabeled and fluorescent OxLDL probes are important tools in the study of scavenger receptor-mediated endocytosis by macrophages and endothelial cells, as well as the formation of macrophage-derived foam cells, a hallmark of early atherogenesis.

The OxLDL probe is generated by oxidizing the surface lipids of native unmodified LDL by incubating with copper sulfate. Oxidation is monitored during the incubation by measuring the optical density at 234 and terminated at approximately the halfway point during the lipid peroxidation phase to a level of approximately 25–35 nmol/mg protein in TBAR (thiobarbituric acid–reactive) values. This controlled oxidation procedure is used to ensure that lipids on the surface of the LDL are oxidized, with limited oxidation of the surface apolipoprotein, allowing the OxLDL to induce a physiologically relevant inflammatory response from cells.

Features of OxLDL:
• Oxidized by copper-mediated process
• Each lot tested for optimal level of oxidation and functionally tested with Bovine Pulmonary Artery Epithelium (BPAE) cells for recognition by scavenger receptors
• Can be used as a negative control to block cell surface receptors or to stimulate inflammatory/immunogenic responses and in applications such as ELISA and Western blot

Oxidized LDL is also complexed with the red-orange fluorescent DiI dye (DiI-OxLDL) to enable fluorescence detection of oxidized LDL uptake. DiI is a highly fluorescent lipophilic dye that diffuses into the hydrophobic portion of the LDL complex and does not affect the LDL-specific binding of the apolipoprotein. The DiI-OxLDL degree of labeling has been optimized for superior sensitivity in various fluorescence applications such as fluorescence microscopy, flow cytometry, cell sorting, high-throughput analysis, and high-content analysis.

Features of DiI-OxLDL:
• Detectable with RFP/TRITC filter set (absorption peak: ∼554 nm, emission peak: ∼571 nm)
• Fixable and multiplexable with green, deep red, violet, and blue emission dyes, such as GFP, DAPI, Hoechst, HCS LipidTOX, and LysoTracker Deep Red
• Each lot functionally tested with Bovine Pulmonary Artery Epithelium (BPAE) cells for recognition by scavenger receptors
• Optimized oxidization and degree of labeling for superior sensitivity