Abstract
Cholesterol efflux (ChE) capacity is associated with the incidence of cardiovascular events and has been proposed as an emerging cardiovascular risk factor. ChE has been traditionally assessed by in vitro radioactive methods but these are not appropriate when assessing a large number of samples. Therefore, alternative, reproducible nonradioactive methods have been developed. This chapter describes a robust nonradioactive method using a fluorescent tracer to assess ChE in vitro.
The measurement of ChE in vitro requires three main components: a cholesterol-loaded donor cell, a cholesterol tracer, and a cholesterol acceptor. This method involves labeling of murine macrophage J774A.1 cells using the fluorescent sterol dipyrromethene boron difluoride (BODIPY)-cholesterol. The cholesterol acceptors from humans or animals include lipid-free apolipoprotein (ApoA)-1, high-density lipoprotein (HDL), HDL2 and HDL3 subfractions, serum, plasma or ApoB-depleted serum or plasma. While lipid-free ApoA-1 mediates ChE via only ATP-binding cassette (ABC)A1 transporter, the remaining acceptors mediate ChE via ABCA1 , ABCG1 and scavenger receptor class B type 1 (SRB1) transporters. The reproducibility of this BODIPY-ChE assay is excellent as the intra-assay coefficients of variation (CVs) were <10% (30 replicates on the same day) and the interassay CVs were <14% (10 experiments performed on different days, with 3 replicates each). The fluorescent method therefore represents a reproducible, safe and useful tool to evaluate ChE as an emerging cardiovascular risk factor.
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Acknowledgments
This work was funded by the AppleCOR Project, which was made possible with the support of the Ministerio de Economía, Indústria y Competitividad, the Agencia Estatal de Investigación, and the European Regional Development Fund. The NFOC-Salut group is a consolidated research group of the Generalitat de Catalunya, Spain (reference no. 2017 SGR 522). The role of the funders was limited to an economic contribution through a competitive call. The funders had no role in the conception, design, performance, or approval of the work.
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Fernández-Castillejo, S., Pedret, A., Catalán Santos, Ú., Solà, R. (2022). A Fluorescence-Based In Vitro Method to Assess Cholesterol Efflux. In: Ramji, D. (eds) Atherosclerosis. Methods in Molecular Biology, vol 2419. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1924-7_15
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DOI: https://doi.org/10.1007/978-1-0716-1924-7_15
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