This work was supported by grants from National Health and Medical Research Council of Australia (NHMRC) (526615 and 545906) and in part by the Victorian Government's OIS Program. DS is a fellow of NHMRC.

1. Preparation of [3H]-cholesterol
<ol>
	<li>In the fume hood, dispense the required amount of [3H]cholesterol into a 1.5 ml microfuge tube (0.5 &mu;Ci (19 kBq) per well is required for a typical assay).</li>
	<li>If [3H]-Cholesterol is suspended in toluene, dry it down completely with N2 gas and resuspend with 100% ethanol to a final concentration of 1 &mu;Ci (37 kBq/&mu;l. Vortex and mix well.</li>
</ol>
2. Plating Cells and Labelling Cellular Cholesterol
This protocol has been tested using the following cell types: human monocytes 4,5,THP-1 human monocyte-macrophages 6,7,8, RAW 264.7 murine macrophages 5,9,10,11, HeLa cells 12, human umbilical vein endothelial cells (HUVEC), BHK-21 cells 9, human and mouse fibroblasts 13,14, HepG2 human hepatocarcinoma cells 15 and, in modified form, from platelets 16 .
<ol>
	<li>Resuspend cells and count them. Plate cells into 12-well plates at the final density of 0.2x106 cells per well in 0.9 ml complete medium. Cells will continue growing and will reach 0.8x106 cells per well by the time of the efflux experiment. For cells that do not divide, such as differentiated THP-1 cells, the seeding density should be 0.8x106 cells per well. If using 6- or 24-well plates, double or half the number of cells per well respectively. The number of wells should be sufficient for quadruplicate determinations for each experimental condition.</li>
	<li>If differentiation of THP-1 cells is required, add PMA (final concentration 0.1 &mu;g/ml) to the media for 48-72 h.</li>
	<li>Add a small aliquot of media into the microfuge tube containing the [3H]cholesterol.</li>
	<li>Wash sides of tube with media before adding [3H]cholesterol into a separate aliquot of complete media (100 &mu;l/well).</li>
	<li>Add the media containing [3H]cholesterol to the wells with cells (final volume per well is 1 ml). If no treatment of cells is required before labelling, cells may be plated in [3H]cholesterol-containing medium.</li>
	<li>Incubate cells for 48 hours in cell culture incubator (37 &deg;C, 5% CO2).</li>
</ol>
3. Equilibration Incubation
<ol>
	<li>After 48 hour incubation, check cells under microscope. Ensure they are healthy and are at approximately 80% confluence.</li>
	<li>Remove media containing [3H]cholesterol. Wash cells gently with PBS. Repeat 3 washing times.</li>
	<li>Prepare serum-free media. If required, activate cells by adding LXR agonist (such as TO-901317 at 1-4 &mu;Mol/L) or cAMP (0.3 mMol/L; for cells of mouse origin only) to serum free-media.</li>
	<li>Add 500 &mu;l serum free media to each well.</li>
	<li>Incubate for 18 hours in the cell culture incubator (37 &deg;C, 5% CO2).</li>
</ol>
4. Cholesterol Efflux Incubation
<ol>
	<li>After 18 hour incubation in serum-free medium, check cells under microscope. Ensure they are healthy and confluent (minimum 80% confluency).</li>
	<li>Prepare solution of cholesterol efflux acceptors in serum-free medium. Examples of acceptors include : 
	Apolipoprotein A-I (apoA-I) ( final concentration 10 &mu;g/ml) 
	High density lipoprotein (HDL)( final concentration 20 &mu;g/ml) 
	Cyclodextrin (final concentration 200 &mu;g/ml) 
	Plasma (final concentration 1-2%)</li>
	<li>Wash cells gently with PBS.</li>
	<li>Add 250 &mu;l of serum-free media with acceptors to each well.</li>
	<li>Leave one set of wells to determine background efflux (efflux to serum free media with NO acceptor)</li>
	<li>Incubate cells for 2 hours in cell culture incubator (37 &deg;C, 5% CO2). Duration of the efflux incubation may vary from 30 min to 8 h if required.</li>
</ol>
5. Processing Samples
<ol>
	<li>After 2 hours incubation check cells under microscope.</li>
	<li>Collect media into 1.5 ml microfuge tubes. Spin at 14,000 rpm for 1-10 min at room temperature to remove cellular debris.</li>
	<li>Transfer 100 &mu;l media into 7 ml scintillation vial. Add 5 mls of Insta-gel Plus (PerkinElmer) and vortex mixture. Store remaining samples at 4 &deg;C.</li>
	<li>Place plates in freezer for 30 mins. Add 500 &mu;l dH2O to each well. Check cells under microscope to ensure that all cells have lifted from the bottom of wells. If still adherent, either leave plates with dH2O at 4 &deg;C overnight or scrape wells.</li>
	<li>Once all cells have lifted off, pipette up and down to break up cell clumps. Transfer 100 &mu;l into 7 ml scintillation vials. Add 5 mls of Insta-gel Plus (PerkinElmer) and vortex mixture. Store remaining plates at 4 &deg;C.</li>
	<li>Place scintillation vials on scintillation counter. Configure counter to count for [3H] in dpm units.</li>
</ol>
6. Analysing the Results
<ol>
	<li>The rate of cholesterol efflux is usually expressed as a proportion of cholesterol moved from cells to the acceptor. The following formula is used:</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/3810/3810eq1.jpg" />
<ol start="2">
	<li>The specific efflux is calculated as a difference between the efflux in the presence or absence of the acceptor (blank).</li>
</ol>
<img alt="Equation 2" src="/files/ftp_upload/3810/3810eq2.jpg" />
7. Timeline
<img alt="Figure 1" src="/files/ftp_upload/3810/3810timeline.jpg" />
8. Representative Results
An example of an outcome of a cholesterol efflux experiment is shown in Fig. 1. In this experiment THP-1 human monocytes were differentiated into macrophages and cholesterol efflux to different acceptors was tested. Cholesterol efflux to medium with no acceptors ("blank") was 0.79% and this value was regarded as a non-specific efflux and was subtracted from other values. Cholesterol efflux to human apoA-I (final concentration 30 &mu;g/ml) was 4.75%. A reference plasma sample was included into this experiment to monitor inter-experimental variability. A reference sample can be used for normalization of data across a large number of experiments, but we found it prudent to repeat the assay if variability is high. A patient plasma (final concentration 2%) was tested before and after the patient was treated with medication. It was concluded that in this patient medication had a negative impact on the capacity of plasma to support cholesterol efflux.
Another example of an outcome of the efflux experiment is shown in Fig. 2. In this experiment RAW 264.7 macrophages were activated or not activated by overnight incubation with LXR agonist TO-901317 (final concentration 1 &mu;mol/L) and cholesterol efflux to the same sample of human plasma (2%) was tested. It was concluded that activation of cellular expression of ABC transporters with LXR agonist increases the capacity of cells to release cholesterol to extracellular acceptor.
<img alt="Figure 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> 
Figure 1. Cholesterol efflux from THP-1 cells to various acceptors. Percentage of cholesterol efflux (i.e. the proportion of labelled cholesterol moved from cells to the specified acceptor) is shown after subtraction of blank values. Mean &plusmn; SD of quadruplicate determinations, *p&lt;0.05.
<img alt="Figure 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> 
Figure 2. Cholesterol efflux from RAW 264.7 activated or not activated with LXR agonist to human plasma. Percentage of cholesterol efflux (i.e. the proportion of labelled cholesterol moved from cells to the specified acceptor) is shown after subtraction of blank values. Mean &plusmn; SD of quadruplicate determinations, *p&lt;0.001.

<ol>
	<li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+in+health+and+disease.">Cholesterol in health and disease.</a> J. Clin. Invest. 110, 583-590 (2002).</li><li>Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+feedback:+from+Schoenheimer's+bottle+to+Scap's+MELADL.">Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL.</a> J. Lipid Res. 50, 15-27 (2009).</li><li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+cellular+cholesterol+accumulation:+basic+concepts+and+physiological+implications.">Consequences of cellular cholesterol accumulation: basic concepts and physiological implications.</a> J. Clin. Invest. 110, 905-911 (2002).</li><li>Murphy, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Density+Lipoprotein+Reduces+the+Human+Monocyte+Inflammatory+Response.">High-Density Lipoprotein Reduces the Human Monocyte Inflammatory Response.</a> Arterioscler. Thromb. Vasc. Biol. 28, 2071-2077 (2008).</li><li>Mujawar, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Immunodeficiency+Virus+Impairs+Reverse+Cholesterol+Transport+from+Macrophages.">Human Immunodeficiency Virus Impairs Reverse Cholesterol Transport from Macrophages.</a> PLoS Biology. 4, 365-36 (2006).</li><li>Patel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Increases+Plasma+High-Density+Lipoprotein+Anti-Inflammatory+Properties+and+Cholesterol+Efflux+Capacity+in+Patients+With+Type+2+Diabetes.">Reconstituted High-Density Lipoprotein Increases Plasma High-Density Lipoprotein Anti-Inflammatory Properties and Cholesterol Efflux Capacity in Patients With Type 2 Diabetes.</a> J. Am. Coll. Cardiol. 53, 962-971 (2009).</li><li>Shaw, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+reconstituted+high-density+lipoprotein+leads+to+acute+changes+in+human+atherosclerotic+plaque.">Infusion of reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic plaque.</a> Circ. Res. 103, 1084-1091 (2008).</li><li>Kekulawala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+freezing+on+high-density+lipoprotein+functionality.">Impact of freezing on high-density lipoprotein functionality.</a> Anal. Biochem. 379, 75-77 (2008).</li><li>D'Souza, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure/Function+Relationships+of+Apolipoprotein+A-I+Mimetic+Peptides:+Implications+for+Antiatherogenic+Activities+of+High-Density+Lipoprotein.">Structure/Function Relationships of Apolipoprotein A-I Mimetic Peptides: Implications for Antiatherogenic Activities of High-Density Lipoprotein.</a> Circ. Res. 107, 217-227 (2010).</li><li>Tchoua, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cholesteryl+ester+transfer+protein+overexpression+and+inhibition+on+reverse+cholesterol+transport.">The effect of cholesteryl ester transfer protein overexpression and inhibition on reverse cholesterol transport.</a> Cardiovasc Res. 77, 732-739 (2008).</li><li>Mukhamedova, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+apolipoprotein+A-I-dependent+cholesterol+efflux+elevates+cholesterol+export+from+macrophages+in+vivo.">Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo.</a> J. Lipid Res. 49, 2312-2322 (2008).</li><li>Mukhamedova, N., Fu, Y., Bukrinsky, M., Remaley, A. T., Sviridov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Different+Regions+of+ATP-Binding+Cassette+Transporter+A1+in+Cholesterol+Efflux.">The Role of Different Regions of ATP-Binding Cassette Transporter A1 in Cholesterol Efflux.</a> Biochemistry. 46, 9388-9398 (2007).</li><li>Sviridov, D., Fidge, N., Beaumier-Gallon, G., Fielding, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+A-I+stimulates+the+transport+of+intracellular+cholesterol+to+cell-surface+cholesterol-rich+domains+(caveolae.">Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae.</a> Biochem. J. 358, 79-86 (2001).</li><li>Smyth, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+harlequin+ichthyosis+delineates+a+key+role+for+abca12+in+lipid+homeostasis.">A mouse model of harlequin ichthyosis delineates a key role for abca12 in lipid homeostasis.</a> PLoS Genet. 4, e1000192-e1000192 (2008).</li><li>Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Caveolin-1+Enhances+Cholesterol+Efflux+in+Hepatic+Cells.">Expression of Caveolin-1 Enhances Cholesterol Efflux in Hepatic Cells.</a> J. Biol. Chem. 279, 14140-14146 (2004).</li><li>Calkin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Attenuates+Platelet+Function+in+Individuals+With+Type+2+Diabetes+Mellitus+by+Promoting+Cholesterol+Efflux.">Reconstituted High-Density Lipoprotein Attenuates Platelet Function in Individuals With Type 2 Diabetes Mellitus by Promoting Cholesterol Efflux.</a> Circulation. 120, 2095-2104 (2009).</li></ol>

The authors declare no conflicting interests related to this study.

The described method to measure cholesterol efflux is designed to measure movement of cholesterol from cells to an extracellular cholesterol acceptor. There are several critical considerations in understanding this methodology.
Labelling and equilibration
In the described methodology labelled cholesterol is added to serum-containing serum. Although never investigated in detail, it is assumed that cholesterol is incorporated into serum lipoproteins and they are taken up by cells. It is important to allow sufficient time for lipoproteins to be taken up and for cholesterol to move from lipoproteins to cellular membranes. Usually 24 h labelling is sufficient, but labelling for 48 h achieves higher specific activity of intracellular cholesterol. It needs to be taken into account that if [14C]cholesterol is used instead of [3H]cholesterol (e.g. in double labelling experiments), the specific activity of the former is very low and adding enough labelled cholesterol to achieve intended specific activity may change cell cholesterol content and thus influence the outcome. It is also important that (i) there is no labelled cholesterol or lipoproteins non-specifically trapped on the cell surface as this cholesterol will be rapidly incorporated by an acceptor via non-specific transfer; and (ii) labelled cholesterol is equilibrated between various intracellular pools. Although in our experience 24 h equilibration incubation in serum-free medium is sufficient to achieve these goals, usually 48 h incubation is used. Many cell types do not survive for 48 h or even 24 h in serum-free medium. In this case 0.1% BSA (essentially fatty-acid free) can act as substitute. Lipoprotein-depleted serum or various serum-replacement supplements can be used as a last resort, but it is important to recognize that lipoprotein-deficient serum does and serum-replacement supplements may contain apolipoproteins and that may affect the outcome. Unfortunately manufacturers often do not disclose the composition of the supplements.
Duration of the efflux incubation
Cholesterol efflux leads to loading of cholesterol to an extracellular acceptor and depletion of cholesterol in cells. Changes in cholesterol content of both cells and acceptor may affect the rate and other properties of cholesterol efflux. Furthermore, if the acceptor contains cholesterol (e.g. HDL) it may move from acceptor to cells and if specific activity of cholesterol in the acceptor becomes significant, that will complicate the interpretation of the results. Thus, it is best to keep efflux incubation as short as possible, allowing however time for sufficient amount of labelled cholesterol to move from cells to the acceptor to enable a reliable detection. Generally 2-6 h is the recommended time. Incubations longer than 24 h would reflect a state of equilibrium and therefore will be indicative of the capacity of acceptor to accumulate cholesterol rather than the rate of cholesterol efflux.
Acceptor
Different acceptors are specific for different pathways of cholesterol efflux. For measuring specific cholesterol efflux, apoA-I is used to reflect ABCA1-dependent efflux whilst HDL or reconstituted HDL for ABCG1 and SR-B1-dependent pathways. Cyclodextrin is often used to assess non-specific efflux. It is important to include in the experiment a "blank" sample, which contains no acceptor at all, to measure the contribution of non-specific dissociation of cholesterol and cholesterol released from disintegrating dead cells.
Concentration of the acceptor
Cholesterol efflux includes (i) release of cholesterol by cells and (ii) its uptake by an acceptor, both steps can be rate-limiting. If the experimental goal is to investigate changes in the capacity of cells to release cholesterol (e.g. after transfection or knock-down), then the concentration of the acceptor should not be rate limiting. For apoA-I and HDL the concentrations of 30-50 &mu;g/ml are usually non rate-limiting. If the capacity of the acceptor to support cholesterol efflux is the goal of investigation (e.g. capacity of isolated HDL or plasma samples from patients or animals to support cholesterol efflux), then the concentration of the acceptor should be rate-limiting. For apoA-I and HDL this is usually 10 &mu;g/ml, for plasma samples this is usually 1-2%, but a preliminary dose-dependence experiment to find a correct concentration is essential to choose the dose.
Activation of cells
The key elements responsible for specific cholesterol efflux are two ABC transporters, ABCA1 and ABCG1. Both are regulated by LXR and pre-incubation of cells with LXR agonist (most popular compound is TO-901317 available from Sigma) used at concentrations 1-4 &mu;mol/L significantly increases the proportion of specific cholesterol efflux. Cells of mouse origin, such as RAW 264.7 or J774, can also be activated by cAMP (0.3 mMol/L).
Loading of cells with cholesterol
Loading of cells with cholesterol by pre-incubating them with acetylated or oxidized LDL or cyclodextrin-cholesterol complex also stimulates cholesterol efflux. It needs to be kept in mind that loading cells, especially macrophages with cholesterol changes many aspects of their metabolism. Choice of model (cholesterol loaded versus not cholesterol loaded) depends on scientific goal of the study.
Presentation of the results
Usually the results of cholesterol efflux experiment are presented as a percentage of cholesterol released from cells: this eliminates variability from cell number in individual wells and in efficiency of labelling. However, this is only valid if treatment did not affect cholesterol uptake and the efflux in the absence of the acceptor (blank). These two conditions need to be experimentally proven.
Theoretically this method can be used to study efflux of any cellular constituency to an extracellular acceptor. If, unlike cholesterol, such compound is metabolised, then an analytical purification step needs to be included. Further, if the compound is synthesized in the cell, synthetic precursors can be used for labelling. For example, [14C]acetate and [3H]water were used to study the efflux of newly synthesized cholesterol and [14C]cholate for studying the efflux of phospholipids. In this case newly synthesized cholesterol needs to be separated from precursors and intermediates, usually by thin layer chromatography.

cholesterol efflux assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

Cholesterol Efflux Assay | Medicine | Video

Cholesterol Efflux Assay

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

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29.7K Views.  Baker IDI Heart and Diabetes Institute. The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Video: Cholesterol Efflux Assay

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Biochemistry

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Immunology and Infection

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

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.

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.

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

Cholesterol Efflux Assay

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Chapters in this video

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Lipid Exchange Assay in Living Cells

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl^- Channels and Transporters

Using Caco-2 Cells to Study Lipid Transport by the Intestine

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion