This work was funded by a Plaxgen Research grant awarded to SM (PLX-1008). We thank Palo Alto Medical Research Foundation Research Institute for collecting serum samples from atherosclerosis subjects under IRB approval.

1. Preparation of Fluorescent-labeled Cholesterol and Lipid-lowering Drugs
NOTE: Fluorescent-labeled cholesterol aggregates and statins were prepared as described in our previous article21. Please see Table of Materials for details of reagents, imaging flow cytometer, chemistry analyzers, and data analysis software used in this study.
<ol>
	<li>Solubilize fluorescent-labeled lyophilized cholesterol (1 mg, Ex/Em = 495 nm/507 nm) powder in 1 mL of 100% alcohol.</li>
	<li>Centrifuge the sample for 3 min at 2,040 x g and use the supernatants containing soluble fluorescence-labeled cholesterol aggregates in the plaque array assay.</li>
	<li>For the preparation of drugs, individually solubilize the powders (2 mg) of simvastatin, lovastatin, atorvastatin, ezetimibe, and omega-3 fatty acid in 1 ml of 100% alcohol.</li>
	<li>After centrifugation for 3 min at 2,040 x g, use the supernatants containing drugs in the assay.</li>
	<li>Similarly, solubilize individually the powders (2 mg) of fluvastatin, rosuvastatin, niacin, and fibrate in 1 mL of deionized water to make a stock solution of 2 mg/mL.</li>
	<li>After centrifugation for 3 min at 2,040 x g, use the supernatants containing drugs in the plaque array assay.</li>
</ol>
2. Plaque Array Assay for Examining In Vitro Cholesterol Particles Formation
<ol>
	<li>Use chemistry analyzer-1 (see Table of Materials) to set up the plaque array assay for cholesterol particles formation. Perform the assay in a round bottom, low protein binding 96-well plate using the reagents prepared in Section 1. Maintain the final reaction volume in each well at 200 &#181;L and perform all assays in triplicate.
	<ol>
		<li>First, load 194.5 &#181;L of phosphate buffered saline (PBS) to each well.</li>
		<li>To each well, add 2.5 &#181;L (5 &#181;g) of each lipid-lowering drug solution and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate to uniformly mix the drug into solution. No drug is added to the negative control samples.</li>
		<li>Finally, add 2 &#181;L (2 &#181;g) fluorescent-labeled cholesterol aggregate solution to each well and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
		<li>Incubate the plate on a lab shaker for 2 h set at 37 &#176;C and 200 rpm.</li>
		<li>After incubation, acquire the images of particles by imaging flow cytometry.</li>
	</ol>
	</li>
</ol>
3. Capturing Images of Cholesterol Particles Using Imaging Flow Cytometry
<ol>
	<li>Open the data acquisition template to load the correct instrument settings. Click &#34;File&#34;, then select &#34;Open Template&#8230;&#34; and select the template file.</li>
	<li>Click &#34;Flush, Lock, Load&#34; to prepare the instrument for sample loading.</li>
	<li>When the &#34;Load Sample&#34; dialogue box opens, click &#34;OK&#34; to load 50 &#181;L of the samples from each well prepared in Section 2 into the imaging flow cytometer.</li>
	<li>Click &#34;Run|Setup&#34; to view the particles in the imaging area in real time.</li>
	<li>When the particles in the imaging area are in good focus, click &#34;Run|Acquire&#34; to acquire images of each object simultaneously in a high-throughput manner for dark field (side scatter/SSC), bright field (BF), green fluorescence, and yellow fluorescence.</li>
	<li>Repeat steps 3.2 - 3.5 to acquire 5,000 - 10,000 particles from each sample.</li>
	<li>Analyze all raw image files using the image analysis software for determining object fluorescence intensity and morphological variations.
	<ol>
		<li>Click the &#34;Tools&#34; button at the top of the image analysis software, then select &#34;Batch Data Files&#34; from the drop-down menu, which will open the &#34;Batches&#34; window.</li>
		<li>In the &#34;Batches&#34; window, click the &#34;Add Batch&#34; button, which will open the &#34;Define a Batch&#34; window.</li>
		<li>In the &#34;Define a Batch&#34; window, click the &#34;Add Files&#34; button and select the raw image files, click the &#34;Template&#34; button and select the appropriate data analysis template file, then select &#34;OK&#34; and &#34;Submit Batches&#34; to begin processing and analysis of the raw image files.</li>
	</ol>
	</li>
</ol>
4. Plaque Array Assay Based Evaluation of Lipid-lowering Drugs Effect on Purified Cholesterol Particles
<ol>
	<li>As described in step 2.1, perform the cholesterol particles formation assay using purified VLDL, LDL, and HDL lipoproteins/particles. Perform the plaque array assay in a 96-well plate.
	<ol>
		<li>First, load all wells with PBS; use a 200 &#181;L final volume for each well.</li>
	</ol>
	</li>
	<li>For negative control, add 4 &#181;g VLDL, LDL, or HDL proteins/particles individually in each well without drugs and shake the plate for 30 s.
	<ol>
		<li>For examining the lipid-lowering drug effect, add 4 &#181;g VLDL, LDL, or HDL proteins/particles individually to each well and shake the plate for 30 s.</li>
		<li>For wells added with 4 &#181;g VLDL, LDL, or HDL, add 2.5 &#181;L (5 &#181;g) ezetimibe, lovastatin, simvastatin, or niacin solution and shake the plate for 30 s by placing it in chemistry analyzer-1.</li>
		<li>Finally, add 2 &#181;L (2 &#181;g) fluorescence-labeled cholesterol aggregate solution to all wells and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Incubate the plate for 2 h in a lab shaker set at 37 &#176;C and 200 rpm.</li>
	<li>Acquire the samples using imaging flow cytometry following steps 3.1 - 3.5.</li>
	<li>Analyze raw image files using image analysis software as described in step 3.7.</li>
	<li>In the analysis template, use the following gating scheme for identification of cholesterol particles subpopulations.
	<ol>
		<li>On a plot of green channel saturated pixel count (x-axis) and dark field channel saturated pixel count (y-axis), reject objects with one or more saturated pixels in either channel.</li>
		<li>Plot the objects/particles without saturated pixels using green channel intensity (x-axis) and SSC intensity (y-axis): objects fall into one of three regions (VLDL, LDL, HDL) based on their green channel and SSC intensities. 
		NOTE: These gates were created based on data generated from control experiments carried out using purified VLDL, LDL, and HDL particles/proteins without the drug.</li>
	</ol>
	</li>
	<li>To determine the lipid-lowering drug effect for each sample, calculate the total lipoprotein particles concentrations, as well as the percentage of each subpopulation (VLDL, LDL, HDL) as described in step 6.5.
	<ol>
		<li>Calculate the particle concentrations (particles/mL) using the following equation: 
		<img alt="Equation" src="/files/ftp_upload/56596/56596eq1.jpg" /> 
		NOTE: The VLDL, LDL, and HDL gating regions in the fluorescence dot-plot detect ~ 80% of their respective subpopulations, and the remaining ~ 20% of the particles overlap with other gating regions.</li>
	</ol>
	</li>
</ol>
5. Measurement of Lipid Concentrations in the Serum Samples
<ol>
	<li>For determining concentrations of LDL, HDL, cholesterol, and triglycerides by chemistry analyzer-2, use serum samples obtained from age-matched normal and dyslipidemia subjects.</li>
	<li>Define abnormal concentration of LDL, HDL, cholesterol, and triglycerides based on their concentration identified beyond the upper and lower reference values in serum samples.</li>
	<li>Follow the normal reference value recommended by the reagent manufacturer: cholesterol (4 - 200 mg/dL), HDL (3.0 - 65 mg/dL), LDL (4 - 100 mg/dL), and triglycerides (9 - 200 mg/dL).</li>
	<li>Identify serum samples that have an abnormal value as either below the lower reference value or above the higher reference value for cholesterol and triglycerides, and use them for screening in the presence and absence of lipid-lowering drugs.</li>
</ol>
6. Analyzing the Effect of Drugs on Cholesterol Particles Formation in Serum Samples
<ol>
	<li>Use the chemistry analyzer-1 to set up the plaque array assay for cholesterol particles formation in serum samples. Perform the assay in a round bottom, low protein binding 96-well plate. Use a final reaction volume of 200 &#181;L/well and perform all assays in triplicate.</li>
	<li>Prepare the plate by loading reagents in a stepwise manner.
	<ol>
		<li>Prepare the control wells.</li>
		<li>Load 193 &#181;L of PBS to each well.</li>
		<li>Add 2.5% (v/v) patient serum (only one serum sample per well) and shake the plate for 30 s by placing it on chemistry analyzer reaction plate.</li>
		<li>Add 2 &#181;L (2 &#181;g) of fluorescence-labeled cholesterol aggregate solution into each well and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Prepare the wells with drugs.
	<ol>
		<li>Load 191 &#181;L of PBS to each well.</li>
		<li>Add 2.5% (v/v) patient serum (only one serum sample per well) and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
		<li>Add 2 &#181;L ezetimibe, lovastatin, simvastatin or niacin solution (4 &#181;g) to all wells except the negative control wells. Add only one drug per well and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
		<li>Add 2 &#181;L of fluorescence-labeled cholesterol aggregate solution (2 &#181;g) into each well and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Load drugs only after all wells (control and drug-treated) have been loaded with their respective serum samples, and add fluorescence-labeled cholesterol at the end of this step.</li>
	<li>Incubate the plate for 2 h in a lab shaker set at 37 &#176;C and 200 rpm.</li>
	<li>Acquire the samples by imaging flow cytometer following settings described in steps 3.1 - 3.6.</li>
	<li>Use the image analyzing software for batch processing all the image files using the same template as described in steps 3.7.
	<ol>
		<li>Perform the data analysis by drawing gates on the plot as described in steps 4.5 and 4.6.</li>
	</ol>
	</li>
	<li>To determine the lipid-lowering drug effect/response (low, moderate, and high) for each sample, calculate the total lipoprotein particle concentration, as well as the percentage of the total particles comprising of VLDL, LDL, and HDL subpopulations.</li>
	<li>Plot the VLDL, LDL, and HDL populations on a histogram of the object&#39;s bright field image, width subtracted from length (length - width), and gate into either globular or linear regions: objects with (length - width) &#8804;2&#160;&#181;m fall into the globular region for calculating the percentage of globular and linear shaped particles.</li>
	<li>Compare the percentage of globular and linear shaped LDL and HDL cholesterol particles identified for each serum sample incubated with and without each drug. Among triplicate values obtained for each serum sample, accept the percentage of globular and linear shaped LDL and HDL particles that fall within SD &#177;2 range for determination of the drug effect.</li>
</ol>

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Dr. Madasamy received grant support from Plaxgen, Inc and has a competing financial interest. The other authors have no competing financial interests to disclose.

In general, the distribution and functional properties of VLDL, LDL, and HDL cholesterol particles in the blood circulation are mainly determined by metabolic, genetic, epidemiological,&#160;cellular, and plasma factors22,23. In the present study, examining the effects of lipid-modifying drugs in the buffer revealed that highly lipophilic drugs such as ezetimibe, lovastatin, simvastatin, and atorvastatin induced a higher level complexity on the morphology of cholesterol particles compared to the lower level effect observed with highly hydrophilic rosuvastatin and fluvastatin drugs. These results are in good agreement with our previous study describing a non-enzymatic mechanism based effect of statins in modulating LDL and HDL cholesterol particles formation in the buffer and serum samples21. Accordingly, the results from present study revealed a non-enzymatic mechanism of action of ezetimibe, niacin, fibrate, and omega-3 fatty acid drugs that may play a direct role in modulating cholesterol particles formation.It is possible that the interactions between drugs and cholesterol aggregates leads to the assembly of large size cholesterol particles that are 2 - 60 &#181;m2, exhibiting globular and linear strand morphologies.
Besides, the results obtained using purified lipoprotein particles suggest interactions between cholesterol aggregates and plasma factors including VLDL, LDL, and HDL proteins that may alter the compositions and morphological properties of the cholesterol particles. The drug treatment results involving purified lipoprotein particles indicated a higher level drug effect on VLDL particles formation compared to their effect observed on LDL cholesterol particles formation. The lovastatin, simvastatin, and ezetimibe drugs were used as pro-drugs and their doses in the assays may be higher than the physiological concentrations.
Interestingly, screening of serum samples showed variations of drug effect on altering the profiles of VLDL, LDL, and HDL cholesterol particles formation, particularly their effect on the formations of linear shaped LDL and HDL particles. These drugs induced reduction on linear shaped LDL and HDL cholesterol particles formation in both dyslipidemia and age-matched normal serum samples. The drug effects observed on reducing linear shaped particles formation was higher in simvastatin, ezetimibe, lovastatin, and niacin. The identification of cholesterol particles with globular and linear strand morphologies in the normal and dyslipidemia serum samples suggests that particles with similar morphologies may form in in vivo conditions. Previous studies have identified the presence of disc and needle-like cholesterol crystals in the atherosclerotic plaques of human and ApoE-/- and LDLR-/- mice models24,25,26,27,28.
The HDL particles circulating in the blood exist as a heterogeneous mixture and the level of small and large size HDL particles along with functional activity are important factors to exert their cardio-protective effect via the reverse cholesterol transport pathway29,30. Recent studies have highlighted the importance of identifying HDL cholesterol particle subfractions for elucidating their role in multiple biological functions such as cholesterol efflux, anti-inflammation, anti-thrombotic, and anti-oxidative31. In addition, a number of studies have reported the effect of lipid-lowering therapy in increasing a low to moderate level of HDL in the plasma1,5,21. Accordingly, the results from this study provide new insights on morphological features of cholesterol particles. Notably, the detection of a higher level of linear shaped HDL cholesterol particles in the serum samples of dyslipidemia subjects suggests that they may be the reliable biomarker for both diagnosis and evaluating effects of lipid-modifying drugs in patients. However, further investigation is required using large clinical samples to better understand the cholesterol particles with distinct morphologies and their association to CVD.
In the plaque array assay for examining the drug effect on assembly of cholesterol particles, we used 2 &#181;g of fluorescence labeled cholesterol aggregates and 5 &#181;gof each drug because: (1) drugs competitively bind to both fluorescence labeled cholesterol and endogenous lipids present in the serum samples; (2) from each sample, we acquired 5,000 to 10,000 cholesterol particles that are assembled into large sizes and shapes ranging from ~2 - 60 &#181;2; (3) we observed a wide variations of drug response among serum samples incubated with the drugs (doses 300 ng to 5 &#181;g) and ~1 - 5% of them incubated with high doses showed no detectable changes in the profile of cholesterol particles formation; and (4) the interaction between cholesterol aggregates and lipid-lowering drugs is mediated by a non-enzymatic process. Hence, the concentrations of the reagents used in the assay may be higher than their physiological level.
In conclusion, we have successfully demonstrated the advantages of an in vitro imaging method described in this study for determining the effect of a broad spectrum of lipid-lowering drugs on modulating morphology and composition of cholesterol particles. The approach of visualizing and quantifying the morphology of lipid particles by employing a constellation of image analysis algorithms may help both the diagnosis of atherosclerosis and to evaluate outcomes of lipid-lowering therapy in patients.

Numerous clinical studies have demonstrated beneficial effects of the lipid-lowering drugs in reducing plasma levels of low-density lipoproteins (LDL) cholesterol and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol, which prevents both primary and secondary incidences of atherosclerosis-related adverse cardiovascular events1,2,3,4,5. Statins, a group of HMG-CoA reductase enzyme inhibitors, block endogenous cholesterol synthesis in the liver that in turn lead to lower circulating levels of LDL cholesterol in the blood6,7. Likewise, the lipid lowering effect of niacin is mediated by its direct and noncompetitive inhibition of hepatocyte diacylglycerol acyltransferase-2, a key liver enzyme involved in triglyceride synthesis8. Comparatively, ezetimibe reduces plasma level of LDL by limiting absorption of exogenous cholesterol by binding to Niemann-Pick C1-Like 1 (NPC1L1) protein located in the epithelial cells of the small intestine9. Fenofibrate, another lipid-lowering drug, substantially reduces plasma concentrations of triglycerides and also moderately decreases LDL cholesterol through the peroxisome proliferator-activated receptors pathway10. Besides, omega-3 fatty acid is reported to have the anti-atherosclerotic effect due to its ability to lower the plasma levels of LDL11.
The lipid-lowering drugs, in addition to their primary effect on lowering LDL cholesterol, have a number of beneficial pleiotropic effects including enhancing the HDL level, improving endothelial functions, reducing inflammation, and inhibiting platelet aggregations12,13,14. However, the underlying mechanism of these drugs in increasing HDL cholesterol particles and modifying their structural features are not fully understood. Since these medicines are widely prescribed to treat atherosclerosis-related cardiovascular diseases (CVDs), it is essential to further investigate their possible roles in determining morphological features and distribution of the lipid particles. The human plasma lipidome consists of approximately 600 distinct lipids and 22 different molecular types of cholesterols that are present in various sizes, shapes, densities, and compositions15,16,17. Analytical methods such as ultra-centrifugation, NMR, and gradient gel electrophoresis are used to characterize LDL and HDL particles and their subfractions18,19. However, application of these methods is limited to studies aimed at determining the effect of drugs in modulating morphology and assembly of the lipid particles. The flow cytometer based plaque array is a functional biochemical assay developed for detection and visualization of serum derived lipid and amyloid plaque particles20. The advantages of in vitro imaging method described in this study enable identification of lipid-modulating drug effects in altering morphology and distribution of cholesterol particles in buffer and serum samples.

<table><tbody><tr><td>TopFluor fluorescent cholesterol</td><td>Avanti Polar lipids</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>simvastatin (pro-drug)</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>lovastatin (pro-drug)</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>rosuvastatin</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>atorvastatin</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>fluvastatin</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>ezetimibe (pro-drug)</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>Niacin</td><td>MilliporeSigma</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>fibrate</td><td>MilliporeSigma</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>omega-3 fatty acid</td><td>MilliporeSigma</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>purified VLDL proteins/particles</td><td>Lee Bio</td><td></td><td></td></tr><tr><td>purified LDL proteins/particles</td><td>Lee Bio</td><td></td><td></td></tr><tr><td>purified HDL proteins/particles</td><td>Lee Bio</td><td></td><td></td></tr><tr><td>Human age-matched serum</td><td>Dx Biosamples</td><td></td><td></td></tr><tr><td>Human atherosclerosis serum</td><td>Bioserve</td><td></td><td></td></tr><tr><td>Human normal serum</td><td>Stanford Blood center</td><td></td><td></td></tr><tr><td>LDL measurement reagent pack</td><td>Roche Diangostics</td><td></td><td></td></tr><tr><td>HDL measurement reagent pack</td><td>Roche Diangostics</td><td></td><td></td></tr><tr><td>Total cholesterol measurment</td><td>Roche Diangostics</td><td></td><td></td></tr><tr><td>96-well microtitre plates</td><td></td><td></td><td></td></tr><tr><td>Triglycerides measurement</td><td>Roche Diangostics</td><td></td><td></td></tr><tr><td>Amnis Imaging Flow cytometer</td><td>Amnis Inc</td><td></td><td></td></tr><tr><td>IDEAS image analysing software</td><td>Amnis Inc</td><td></td><td></td></tr><tr><td>Chemistry Analyzer-1, ChemWel 2902</td><td>Awarness Technology</td><td></td><td></td></tr><tr><td>Chemistry Analyzer-2, Intergra 400</td><td>Roche Diangostics</td><td></td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles at JoVE.com

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

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

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 ...

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14.5K Views.  Plaxgen Inc. 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.

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

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Environment

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.

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 ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Biochemistry

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 ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

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

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, ...

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

Summary

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Chapters in this video

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Cholesterol Efflux Assay

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Formulation and Characterization of Bioactive Agent Containing Nanodisks

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

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

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