A Reliable, Non-radioactive Method for Monitoring Accumulation of Cholesterol Inside Mammalian Cells After Loading with Low Density Lipoprotein (ldl)
Agents identified as cholesterol inhibitors are useful in preventing and treating cardiovascular and neurodegenerative diseases associated with over accumulation of cholesterol in cells. These agents are expected to slow the development of atherosclerosis and the accumulation of amyloid beta-peptides in the brain, thereby slowing down the symptoms of Alzheimer’s disease. Therefore, methods for detecting and monitoring cellular cholesterol is of critical importance to human health.Dartmouth scholars discovered that by modifying cell fixation conditions, BCθ, a poreforming cytolysin derivative which binds free cholesterol, can be used to detect intracellular cholesterol-rich domains. When the cells were fixed using our method, BCθ became permeable to cells, and mainly stained cholesterol-rich domains inside the cells., with the signal being the strongest in cells constitutively expressing the LDL receptor and various cholesterol biosynthetic enzymes at elevated levels (i.e., CT43 and 25RA cells) . The sensitivity of BCθ staining was superior to that of filipin staining. Clear differences in intracellular staining with BCθ were also seen between normal and mutant NPC1 fibroblasts of human or mouse origin. Additional results show that BCθ is a powerful tool for examining movement and accumulation of LDL-derived cholesterol inside mammalian cells.
Since BCθ signal can be monitored using fluorescence instead of radioactivity, the use of BCθ in combination with 25RA and CT43 cells is well-suited for high-throughput rapid screening for compounds that interfere with cholesterol accumulation after loading with LDL.
These findings are claimed in the issued United States Patent No. 7,186,519, and the published United States Patent Application No. 10/534,295. We are seeking an industrial partner who is interested in their further refinement and commercialization. (Ref: J215)
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