High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Summary

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Abstract

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [³H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Introduction

According to the World Health Organization, the current principal causes of death worldwide are ischemic heart disease and stroke (accounting for a total of 15.2 million deaths)¹. Both are cardiovascular diseases (CVD) that can be preceded by atherosclerosis and the rupture of atheroma plaques in the blood vessels^2,3.

Atherosclerosis is a vessel wall inflammatory disease in which macrophages, T cells, mast cells, and dendritic cells infiltrate the endothelium and accumulate from the blood, eventually forming atherosclerotic plaques. Atherosclerotic plaques pr....

Protocol

For this study, ethical committee approval (Comitè Ètic d'Investigació Clínica, Hospital Clinic, Barcelona; approval number HCB/2014/0756) and written, informed consent from all subjects were obtained.

1. NBD-cholesterol Preparation

1. Dissolve the NBD-cholesterol (MW 494.63; see Table of Materials) in pure ethanol to obtain the stock (2 mM). For a 10 mg vial, dissolve the entire vial contents in a 10.1 mL volume of ethanol to obtain a 2 mM stock.
2. Dilute the NBD-cholesterol from the stock in RPMI 1640 supplemented with 10% fetal bovine serum and 5% penicillin/streptomycin (R10 medium) to reach a final con....

Results

The aim of the cholesterol efflux assay is to determine in vitro the cholesterol efflux capacity of a given serum, plasma, or supernatant containing HDL particles. The method consists of loading labelled-cholesterol into a culture of a standard macrophage cell line and inducing contact with the testing sample diluted into FBS-free media with the cells. Finally, the fluorescent levels from the NBD are measured in the media and cell lysate. To optimize measurements, the effluxed cholesterol from the cells to media is mixed.......

Discussion

Fluorescent-labelled cholesterol is a promising strategy to analyze and investigate the properties and metabolism of natural cholesterol in vitro. Its main advantages are that it can be taken up by cells, allows for intracellular and membrane distribution studies, and can be applied to cholesterol efflux assays such as in this protocol (Figure 7). Some fluorescent-labelled sterols allow cholesterol tracking in vitro including BODIPY-cholesterol, dansyl-cholesterol, dehydroegrosterol, and 22-.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well collecting plate	Corning Inc	Costar 3912	white 96-well plate with opaque clear bottom
96-well culture plate	Corning Inc	Costar 3610	white 96-well plate with flat clear bottom
Cholesterol Efflux Assay Kit (Cell-based)	Abcam	ab196985	commercial high-throughput cell-based assay kit aimed to determine the cholesterol efflux
colorless RPMI 1640	Sigma-Aldrich	R7509	RPMI 1640 with no pehnol-red
Gen5 Data Analysis Software	BioTek		Version 2.0
Glycine	Sigma-Aldrich	G8790-100G	Glycine, non-animal use
Luminometer	Biotek	SYNERGY HT	Multi-Detection Microplate Reader
Lysis Solution 1	in-house		50 mM Tris Buffer, 150 mM NaCl and H2O
Lysis Solution 2	in-house		Pure ethanol and Cell Lysis Solution 1:1 (v:v)
NBD-cholesterol	Thermo-Fisher	N1148	22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3β-Ol
PBS	Sigma-Aldrich	P3813	Phosphate-buffered saline
PEG 8000	Sigma-Aldrich	202452-250G	polyethylene glycol
PMA	Sigma-Aldrich	79346	Phorbol 12-merystate B-acetate.
R10	in-house		RPMI 1640 supplemented with 10% fetal bovine serum and 5% Penicillin/Streptomycin.
RPMI 1640	Sigma-Aldrich	R8758	RPMI 1640 with 2 mM L-glutamine containing 1.5 g/L sodium bicarbonate and 4.5 g/L glucose
THP-1 cells	Sigma-Aldrich	ATCC, #TIB-202	monocyte-like line derived from leukemia from a one year old baby
Tween 80	Sigma-Aldrich	P1754