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 present a lipid core and cholesterol crystals, evidenced by high-resolution B-mode ultrasonography measurements of the carotid intima media thickness^4,5. In macrophages, cholesterol efflux towards lipid acceptor particles is carried out by means of the ATP-binding cassette (ABC) receptors ABCA1, ATP binding cassette subfamily G member 1 (ABCG1), and the scavenger receptor SR-BI. The imbalance of cholesterol influx and efflux in macrophages is considered a key process in atherosclerosis initiation⁶. Cholesterol efflux is considered a key step in cholesterol elimination from peripheral tissue to the plasma and liver in a process called reverse cholesterol transport (RCT). Cholesterol is transferred from macrophages mainly to apolipoprotein A1 (ApoA1) found on the surface of high-density lipoprotein (HDL) particles. HDLs then transport cholesterol to the liver for excretion and re-utilization^7,8,9.

Traditionally, tritium (³H) radio-labelled cholesterol has been used in cholesterol efflux¹⁰. The emission signal of radioisotopes is highly sensitive¹⁰; however, radio-labelled cholesterol presents obvious handicaps such as long protocols, risk of exposure to ionizing radiation, and the need for special radioactivity facilities and equipment to ensure safe handling of radioactive emission. On the contrary, fluorescence has been successfully incorporated in diagnostic techniques due to its simplicity in fluorescent signal detection, the wide variety of fluorophores available, and its safety¹¹. Several fluorescent-labelled sterols have been used to study cholesterol metabolism including dehydroergosterol (with intrinsic fluorescence), dansyl cholesterol, 4,4-difluoro-3a,4adiaza-s-indacene (BODIPY)-cholesterol, and 22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3β-Ol (NBD-cholesterol). Particularly, NBD-cholesterol presents an efficient uptake in human cells¹². Two different NBD labelled-cholesterol are currently available: 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (22-NBD) and 25-(N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino)-27-norcholesterol (25-NBD; Figure 1). Cholesterol labelled with 22-NBD moiety may best suit cholesterol efflux studies, while 25-NBD-cholesterol is mainly used in cellular membrane dynamics research^13,14.

Cell lines typically used in in vitro cholesterol efflux assays are monocyte-like cells such as human leukemia-derived THP-1 cells, murine Raw 264.7 cells¹⁵, or J774.1. All of these cells can be differentiated into macrophages in vitro using phorbol 12-myristate 13-acetate (PMA), but THP-1-derived macrophages (dmTHP-1) best reflect and mimic the human macrophages¹⁶.

In the present study, we optimize and standardize a fluorescent high-throughput method to determine the cholesterol efflux capacity of serum samples on dmTHP-1, using 22-NBD-cholesterol as an alternative to [³H]-cholesterol. In addition, we compare the optimized fluorescent technique with the standard radioactive analog.

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 concentration of 5 μM (e.g., obtain 10 mL of NBD-cholesterol at 5 μM final concentration) by diluting 25 μL of NBD-cholesterol stock (2 mM) in R10 medium. This volume is sufficient for a 96-well plate.

2. Cell Culture and Seeding (Day-2)

1. Culture THP-1 cells in R10 medium at 37 °C, 5% CO₂. Adjust to 0.3 x 10⁶ cells/mL every 3 days.
2. Seed the cells in a white 96-well plate with a flat, clear bottom at 0.2 x 10⁶ cells/well in R10 medium (100 μL per well).
3. Treat the cells with 100 nM phorbol 12-myristate 13-acetate (PMA; from a 10 μM stock) for 48–72 h, incubating at 37 °C, 5% CO₂ to differentiate THP-1 cells into dmTHP-1.
   NOTE: It is recommended to use 5 μL of PMA stock (10 μM) in 500 μL of R10 medium. Prior to this, prepare a 10 μM PMA stock by dissolving a lyophilized 1 g vial in 10 mL of dimethyl sulfoxide (DMSO), aliquot, and stock at -20 °C.
4. Alternatively (suggestion) combine steps 2.2 and 2.3 for better homogenization. Prepare 10 mL of THP-1 cells in a 15 mL tube, add 200 μL of PMA stock (10 μM), mix gently, and immediately seed 100 μL per well onto the plate. Incubate the cells as described in step 2.3.

3. Apolipoprotein B Depleted Serum (ABDS) Preparation (Day 2 or 3)

1. To prepare a polyethylene glycol (PEG) solution, dilute glycine in 10% PBS (with sterile H₂O) to a concentration of 200 mM at pH 7.4. Add 40 mL of 200 mM glycine to 10 g of PEG 8000 to obtain PEG 20% (w/v). Mix the solution vigorously to homogenize.
2. Apply 4 parts of 20% PEG per 10 parts of serum/plasma on each serum/plasma sample in a 1.5 mL tube and leave the mixture on ice for 25 min¹⁷ (e.g., for 100 μL of plasma or serum, add 40 μL of 20% PEG solution).
3. Centrifuge the PEG-apolipoprotein B precipitate at 13,000 x g for 15 min at 4 °C.
4. Discard the precipitate. Transfer the supernatant to a new tube.
   NOTE: We suggest preparing the ABDS on day 3 (fresh). If it is not possible, prepare the ABDS on day 2 and keep it at 4 °C overnight.

4. NBD-cholesterol Cell Loading (Day 2)

1. Discard the culture medium of the dmTHP-1 and wash the cells twice with 1x phosphate-buffered saline (PBS).
2. Load the cells with 5 μM NBD-cholesterol (100 μL per well) in R10 medium and incubate overnight at 37 °C, 5% CO₂.

5. Incubation with Cholesterol Acceptors (Day 3)

1. Discard the medium and wash the cells twice with PBS.
2. Incubate the cells with 2–5% ABDS or the desired concentration of purified lipid acceptor (HDL, ApoA, ApoE, etc.) diluted in colorless RPMI 1640 medium (100 μL per well) for 4–6 h at 37 °C.
   NOTE: Include a negative control (C-) consisting of colorless RPMI 1640 medium without acceptors and a positive control (C+) (e.g., ABDS from a pool of healthy donors or purified HDLs). Note that the positive control applies particularly when patient samples (disease conditions) are analyzed (Supplementary Figure 1).
3. Prepare a 200 mL stock of the cell lysis solution 1 (50 mM Tris buffer, 150 mM NaCl, H₂O). Mix the cell lysis solution 1 at 1:1 (v:v) ratio with pure ethanol to obtain the lysis solution 2.

6. Fluorescent Signal Capture (Day 3)

1. Media NBD-cholesterol detection
   1. Remove the cell medium from the plates and collect it in a new white 96-well plate with an opaque flat bottom.
   2. For an optimal fluorescence signal detection in the media samples, add 100 μL of pure ethanol to 100 μL of each medium sample to obtain a 1:1 ratio in the 96-well white plate.
   3. Keep the plate with the treated media to further measure the fluorescence intensity (FI) at a 463⁄536 nm (excitation/emission) wavelength in the luminometer.
2. Intracellular NBD-cholesterol detection
   1. Wash the cells twice with PBS.
   2. To obtain the intracellular cholesterol, lyse the cells by incubating them with 100 μL of lysis solution 2 per well and shake the plate at room temperature (RT) for 25 min.
   3. For an optimal fluorescence signal detection in the cell lysate samples, capture the fluorescence intensity of the cell lysates in the same white 96-well plate with clear flat bottom (the same plate in which cells were seeded in step 2.2).
3. 6.3 Measure the fluorescence intensity (FI) in the luminometer while adjusting the sensitivity parameter to 50 in the software (see Table of Materials).

7. Results Analysis

1. Express the cholesterol efflux rate of a sample as a percentage calculated by the formula:
   
2. Obtain the final measure of cholesterol efflux (CE) by subtracting the CE of the negative control (sample loaded with NBD-cholesterol but incubated with colorless RPMI 1640 medium, without cholesterol acceptors) from the CE of a given samples:
   

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