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.

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	<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. 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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. 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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...

<table border="1">
 <tbody>
 <tr>
 <td>Reagents\Cells</td>
 <td>HeLa</td>
 <td>THP-1</td>
 <td>RAW</td>
 <td>HUVEC</td>
 <td>BHK-21</td>
 <td>HFF</td>
 </tr>
 <tr>
 <td>Complete Media</td>
 <td colspan="3">RPMI 
 - 10% FCS 
 - 1% L-glutamine 
 - 0.2% Pen strep </td>
 <td>M199 
 - 5% FCS 
 - 1% L-glutamine 
 - 1% Pen strep 
 - 2% HEPES (1M) 
 - 7% Endothelial cell growth supplement (ECGS) </td>
 <td colspan="2">DMEM 
 - 10% FCS 
 - 1% L-glutamine 
 - 0.2% Pen strep </td>
 </tr>
 <tr>
 <td>Serum Free Media</td>
 <td colspan="3">RPMI 
 - 1% L-glutamine 
 - 0.2% Pen strep </td>
 <td>M199 
 - 1% L-glutamine 
 - 1% Pen strep 
 - 2% HEPES (1M) 
 - 7% Endothelial cell growth supplement (ECGS) </td>
 <td colspan="2">DMEM 
 - 1% L-glutamine 
 - 0.2% Pen strep </td>
 </tr>
 <tr>
 <td>Passage Method </td>
 <td>1 x Trypsin</td>
 <td>Suspension</td>
 <td>Scrape</td>
 <td colspan="3">1 x Trypsin</td>
 </tr>
 <tr>
 <td>Passage Ratio</td>
 <td>3:10 </td>
 <td>1:3</td>
 <td></td>
 <td>1:20 </td>
 <td>1:3</td>
 <td>1:3</td>
 </tr>
 <tr>
 <td>Cell Activation (optional)</td>
 <td>&nbsp;</td>
 <td colspan="5">TO-901317 
 - LXR-agonist 
 - Final conc. 4 &mu;M </td>
 </tr>
 <tr>
 <td>Other Reagents</td>
 <td>&nbsp;</td>
 <td>phorbol 12-myristate 13-acetate (PMA) 
 - Cell differentiation 
 - Final conc. 0.1&mu;g/ml </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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

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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Video: Cholesterol Efflux Assay

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This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

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In Vivo Canine Muscle Function Assay

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In Vivo Canine Muscle Function Assay

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Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

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An in vivo Assay to Test Blood Vessel Permeability

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An in vivo Assay to Test Blood Vessel Permeability

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The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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.