A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Sankaranarayanan, Sandhya; Kellner-Weibel, Ginny; Llera-Moya, Margarita de la; Phillips, Michael C.; Asztalos, Bela F.; Bittman, Robert; Rothblat, George H.

doi:10.1194/jlr.d018051

Cited by 179 publications

(179 citation statements)

References 37 publications

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“…Mouse macrophage-derived J774 cells were maintained in RPMI containing 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin. ABCA1-mediated efflux from HEK293 or J774 cells was determined as described with slight modifications (43,44). HEK293 cells were plated in 24-well collagen-coated plates.…”

Section: Cholesterol Efflux Assaymentioning

confidence: 99%

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Mei

Liu

Herscovitz

et al. 2016

Journal of Lipid Research

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Section: Cholesterol Efflux Assaymentioning

confidence: 99%

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Mei

Liu

Herscovitz

et al. 2016

Journal of Lipid Research

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“…This fluorescent sterol exhibits good correlation with radioisotope-labeled cholesterol in the efflux assay in THP-1 cells (24). Sankaranarayanan et al demonstrated that another fluorescent sterol, bearing a BODIPY fluorophore, can be used as a sensitive probe in the cholesterol efflux assay in J774 mouse macrophages (11). NBD-cholesterol is a commercially available and widely used fluorescent analog of cholesterol, which may be used in cholesterol efflux assays (25)(26)(27).…”

Section: Discussionmentioning

confidence: 99%

“…For decades, fluorescent cholesterol analogs have been used in membrane biophysics for the investigation of lipid trafficking and the membrane organization of native cholesterol (8,9). Until recently, fluorescent sterols were also used in cholesterol efflux assays as an alternative for radioisotope-labeled cholesterol (10,11). Commonly used fluorescent analogs of cholesterol include N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-23,24-bisnor-5-xholen-3β-ol (NBD)-cholesterol, boron-dipyrromethene (BODIPY)-cholesterol and dehydroergosterol with intrinsic fluorescence (8).…”

Section: Introductionmentioning

confidence: 99%

“…Thus, these findings support NBD-cholesterol as a sensitive and specific probe, which may be used efficiently for cholesterol efflux measurement. Several other studies have reported that fluorescent BODIPY-cholesterol and Pennsylvania Green-cholesterol can also be used in cholesterol efflux assay as substitutes for [ 3 H]-cholesterol (11,24). Further investigation to compare different fluorescent analogs in parallel with [ 3 H]-cholesterol may assist in determining which fluorescent analog can mimic native cholesterol efflux most closely in the assay.…”

mentioning

confidence: 99%

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Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages

Song

Wang

et al. 2015

Molecular Medicine Reports

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Abstract. Macrophage cholesterol efflux is important in maintaining cellular lipid homeostasis and preventing the formation of lipid-laden foam cells. Although radioactive [ 3 H]-cholesterol is widely used as a tracer in cholesterol efflux assays, the lengthy and labor-intensive assay procedure, and the radioactivity disposal procedure limit the use of this assay for high-throughput screening. In the present study, a novel procedure using fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-23,24-bisnor-5-xholen-3β-ol (NBD)-cholesterol was developed as a substitute for [ .872 for apoA-1; P<0.001). Furthermore, NBD-cholesterol efflux in the THP-1 cells exhibited a similar trend to that obseved in the PBMCs. In conclusion, the results of the present study suggested that fluorescent NBD-cholesterol can be used as a sensitive and specific probe in cholesterol efflux assays in THP-1-derived macrophages. IntroductionCoronary artery disease (CAD) is the leading contributor to morbidity and mortality rates worldwide, and results in a mortality rate of >7,000,000 per year (1). Atherosclerotic plaques are key in the pathophysiology of CAD, in which macrophages initially become foam cells and initiate plaque formation. The imbalance of cholesterol uptake and efflux in macrophages may cause excessive accumulation of cholesterol and foam cell formation, which is a hallmark in the development of atherosclerosis (2). Cholesterol efflux from macrophages is a critical step in reverse cholesterol transport, which may prevent macrophages from becoming foam cells and thus reduce atherosclerosis (3,4). Free cholesterol is transported from macrophages in the arterial atherosclerotic plaque to an extracellular acceptors, including apoliporotein (apo) A-1 and high-density lipoprotein (HDL), and then back to the liver for bile acid synthesis and excretion (5). Previously, Khera et al reported a reverse correlation between cholesterol efflux capacity and atherosclerosis (6). To understand the underlying mechanism regulating cholesterol efflux, it is important to develop a reliable assay for measuring cholesterol efflux in vitro.Traditionally, radioisotope-labeled cholesterols have been used as probes in cholesterol efflux assays. However, the procedure is lengthy and labor-intensive, and the radioactivity disposal procedure can limit the use of this assay in high-throughput screening. By contrast, fluorescent

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“…Isolated primary rat adipose cells were incubated with a BODIPY-cholesterol solution in MEM [0.05 mM BODIPY-cholesterol "Topfl uor" (Avantipolar lipids)], 0.2 mM unlabeled cholesterol, and 10 mM methyl-B-cyclodextrin (m ␤ CD), prepared according to ( 33 ) for 1 h at 37°C. The ratio of cells to BODIPYcholesterol solution was 1:2 (v/v), with a fi nal concentration of 1% BSA (w/v) and 200 nM adenosine.…”

Section: Cholesterol Effl Uxmentioning

confidence: 99%

ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation

Lindahl

Petrlová

Dalla-Riva

et al. 2015

Journal of Lipid Research

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A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Cited by 179 publications

References 37 publications

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages

ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation

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