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High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay
Published on: January 7, 2019
A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol
Sandhya Sankaranarayanan1, Ginny Kellner-Weibel1, Margarita de la Llera-Moya1
1Department of Pediatrics (Division of Gastroenterology, Hepatology, and Nutrition), The Children's Hospital of Philadelphia, Philadelphia, PA 19104.
Journal of Lipid Research
|September 30, 2011
Summary
This study introduces BODIPY-cholesterol as a superior fluorescent probe for measuring cellular cholesterol efflux, offering a more efficient and sensitive high-throughput assay for coronary artery disease (CAD) research compared to traditional radioactive methods.
Area of Science:
- Biochemistry
- Cardiovascular Disease Research
- Assay Development
Background:
- Cellular cholesterol efflux is inversely associated with coronary artery disease (CAD).
- Traditional methods using tritium-labeled cholesterol ([3H]cholesterol) are not suitable for high-throughput screening.
- A novel fluorescent sterol probe is needed to facilitate large-scale studies on cholesterol efflux and CAD.
Purpose of the Study:
- To compare the efficacy of BODIPY-cholesterol with [3H]cholesterol for measuring cellular cholesterol efflux.
- To evaluate BODIPY-cholesterol as a sensitive probe for high-throughput screening in CAD research.
Main Methods:
- J774 macrophages were labeled with either [3H]cholesterol or BODIPY-cholesterol.
- Cholesterol efflux was measured using various acceptors, including apo A-I, HDL(3), and apoB-depleted human serum.
- Correlation and reproducibility analyses were performed to compare the two methods.
Main Results:
- BODIPY-cholesterol showed significantly higher fractional efflux compared to [3H]cholesterol.
- BODIPY-cholesterol efflux strongly correlated with [3H]cholesterol efflux in apoB-depleted sera.
- The BODIPY-cholesterol assay demonstrated excellent reproducibility and sensitivity for ABCA1-mediated efflux.
Conclusions:
- BODIPY-cholesterol offers a more efficient and sensitive method for measuring cholesterol efflux than [3H]cholesterol.
- This fluorescent probe enables a high-throughput assay suitable for screening large numbers of sera.
- The assay facilitates further investigation into the relationship between cholesterol efflux and atherosclerosis.
