Name: high-throughput nitrobenzoxadiazole-labeled cholesterol efflux assay
Uploaded: 2019-01-07T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay at JoVE.com

This work has been partially supported by the research grants FIS (PS12/00866) from Instituto de Salud Carlos III, Madrid, Spain; Fondo Europeo para el Desarrollo Regional (FEDER); Red de Investigaci&#243;n en SIDA (RIS), ISCIII-RETIC (RD16/0025/0002) and CERCA Programme / Generalitat de Catalunya. The authors thank the Retrovirology and Viral Immunopathology Laboratory of the Institut d&#39;Investigacions Biom&#232;diques August Pi I Sunyer (IDIBAPS). We thank T. Escrib&#224;, C. Rovira, and C. Hurtado for their assistance and S. Cuf&#237; from the Knowledge and Technology Transfer Office for her guidance in protecting the invention.

For this study, ethical committee approval (Comit&#232; &#200;tic d&#39;Investigaci&#243; Cl&#237;nica, Hospital Clinic, Barcelona; approval number HCB/2014/0756) and written, informed consent from all subjects were obtained.
1. NBD-cholesterol Preparation
<ol>
	<li>Dissolve the NBD-cholesterol (MW 494.63; see Table of Materials) in pure ethanol to obtain the stock (2 mM). For a 10 mg vial, dissolve the entire vial contents in a 10.1 mL volume of ethanol to obtain a 2 mM stock.</li>
	<li>Dilute the NBD-cholesterol from the stock in RPMI 1640 supplemented with 10% fetal bovine serum and 5% penicillin/streptomycin (R10 medium) to reach a final concentration of 5 &#956;M (e.g., obtain 10 mL of NBD-cholesterol at 5 &#956;M final concentration) by diluting 25 &#956;L of NBD-cholesterol stock (2 mM) in R10 medium. This volume is sufficient for a 96-well plate.</li>
</ol>
2. Cell Culture and Seeding (Day-2)
<ol>
	<li>Culture THP-1 cells in R10 medium at 37 &#176;C, 5% CO2. Adjust to 0.3 x 106 cells/mL every 3 days.</li>
	<li>Seed the cells in a white 96-well plate with a flat, clear bottom at 0.2 x 106 cells/well in R10 medium (100 &#956;L per well).</li>
	<li>Treat the cells with 100 nM phorbol 12-myristate 13-acetate (PMA; from a 10 &#956;M stock) for 48&#8211;72 h, incubating at 37 &#176;C, 5% CO2 to differentiate THP-1 cells into dmTHP-1. 
	NOTE: It is recommended to use 5 &#956;L of PMA stock (10 &#956;M) in 500 &#956;L of R10 medium. Prior to this, prepare a 10 &#956;M PMA stock by dissolving a lyophilized 1 g vial in 10 mL of dimethyl sulfoxide (DMSO), aliquot, and stock at -20 &#176;C.</li>
	<li>Alternatively (suggestion) combine steps 2.2 and 2.3 for better homogenization. Prepare 10 mL of THP-1 cells in a 15 mL tube, add 200 &#956;L of PMA stock (10 &#956;M), mix gently, and immediately seed 100 &#956;L per well onto the plate. Incubate the cells as described in step 2.3.</li>
</ol>
3. Apolipoprotein B Depleted Serum (ABDS) Preparation (Day 2 or 3)
<ol>
	<li>To prepare a polyethylene glycol (PEG) solution, dilute glycine in 10% PBS (with sterile H2O) to a concentration of 200 mM at pH 7.4. Add 40 mL of 200 mM glycine to 10 g of PEG 8000 to obtain PEG 20% (w/v). Mix the solution vigorously to homogenize.</li>
	<li>Apply 4 parts of 20% PEG per 10 parts of serum/plasma on each serum/plasma sample in a 1.5 mL tube and leave the mixture on ice for 25 min17 (e.g., for 100 &#956;L of plasma or serum, add 40 &#956;L of 20% PEG solution).</li>
	<li>Centrifuge the PEG-apolipoprotein B precipitate at 13,000 x g for 15 min at 4 &#176;C.</li>
	<li>Discard the precipitate. Transfer the supernatant to a new tube. 
	NOTE: We suggest preparing the ABDS on day 3 (fresh). If it is not possible, prepare the ABDS on day 2 and keep it at 4 &#176;C overnight.</li>
</ol>
4. NBD-cholesterol Cell Loading (Day 2)
<ol>
	<li>Discard the culture medium of the dmTHP-1 and wash the cells twice with 1x phosphate-buffered saline (PBS).</li>
	<li>Load the cells with 5 &#956;M NBD-cholesterol (100 &#956;L per well) in R10 medium and incubate overnight at 37 &#176;C, 5% CO2.</li>
</ol>
5. Incubation with Cholesterol Acceptors (Day 3)
<ol>
	<li>Discard the medium and wash the cells twice with PBS.</li>
	<li>Incubate the cells with 2&#8211;5% ABDS or the desired concentration of purified lipid acceptor (HDL, ApoA, ApoE, etc.) diluted in colorless RPMI 1640 medium (100 &#956;L per well) for 4&#8211;6 h at 37 &#176;C. 
	NOTE: Include a negative control (C-) consisting of colorless RPMI 1640 medium without acceptors and a positive control (C+) (e.g., ABDS from a pool of healthy donors or purified HDLs). Note that the positive control applies particularly when patient samples (disease conditions) are analyzed (Supplementary Figure 1).</li>
	<li>Prepare a 200 mL stock of the cell lysis solution 1 (50 mM Tris buffer, 150 mM NaCl, H2O). Mix the cell lysis solution 1 at 1:1 (v:v) ratio with pure ethanol to obtain the lysis solution 2.</li>
</ol>
6. Fluorescent Signal Capture (Day 3)
<ol>
	<li>Media NBD-cholesterol detection
	<ol>
		<li>Remove the cell medium from the plates and collect it in a new white 96-well plate with an opaque flat bottom.</li>
		<li>For an optimal fluorescence signal detection in the media samples, add 100 &#956;L of pure ethanol to 100 &#956;L of each medium sample to obtain a 1:1 ratio in the 96-well white plate.</li>
		<li>Keep the plate with the treated media to further measure the fluorescence intensity (FI) at a 463&#8260;536 nm (excitation/emission) wavelength in the luminometer.</li>
	</ol>
	</li>
	<li>Intracellular NBD-cholesterol detection
	<ol>
		<li>Wash the cells twice with PBS.</li>
		<li>To obtain the intracellular cholesterol, lyse the cells by incubating them with 100 &#956;L of lysis solution 2 per well and shake the plate at room temperature (RT) for 25 min.</li>
		<li>For an optimal fluorescence signal detection in the cell lysate samples, capture the fluorescence intensity of the cell lysates in the same white 96-well plate with clear flat bottom (the same plate in which cells were seeded in step 2.2).</li>
	</ol>
	</li>
	<li>6.3 Measure the fluorescence intensity (FI) in the luminometer while adjusting the sensitivity parameter to 50 in the software (see Table of Materials).</li>
</ol>
7. Results Analysis
<ol>
	<li>Express the cholesterol efflux rate of a sample as a percentage calculated by the formula: 
	<img alt="Equation" src="/files/ftp_upload/58891/58891eq1.jpg" /></li>
	<li>Obtain the final measure of cholesterol efflux (CE) by subtracting the CE of the negative control (sample loaded with NBD-cholesterol but incubated with colorless RPMI 1640 medium, without cholesterol acceptors) from the CE of a given samples: 
	<img alt="Equation 2" src="/files/ftp_upload/58891/58891eq2.jpg" /></li>
</ol>

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The authors wish to declare that they are inventors of the patent application (EPO; 18382337.6-1118; 17th May 2018) entitled &#34;Method for determining cholesterol efflux&#34; based on this method.

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

According to the World Health Organization, the current principal causes of death worldwide are ischemic heart disease and stroke (accounting for a total of 15.2 million deaths)1. Both are cardiovascular diseases (CVD) that can be preceded by atherosclerosis and the rupture of atheroma plaques in the blood vessels2,3.
Atherosclerosis is a vessel wall inflammatory disease in which macrophages, T cells, mast cells, and dendritic cells infiltrate the endothelium and accumulate from the blood, eventually forming atherosclerotic plaques. Atherosclerotic plaques present a lipid core and cholesterol crystals, evidenced by high-resolution B-mode ultrasonography measurements of the carotid intima media thickness4,5. In macrophages, cholesterol efflux towards lipid acceptor particles is carried out by means of the ATP-binding cassette (ABC) receptors ABCA1, ATP binding cassette subfamily G member 1 (ABCG1), and the scavenger receptor SR-BI. The imbalance of cholesterol influx and efflux in macrophages is considered a key process in atherosclerosis initiation6. Cholesterol efflux is considered a key step in cholesterol elimination from peripheral tissue to the plasma and liver in a process called reverse cholesterol transport (RCT). Cholesterol is transferred from macrophages mainly to apolipoprotein A1 (ApoA1) found on the surface of high-density lipoprotein (HDL) particles. HDLs then transport cholesterol to the liver for excretion and re-utilization7,8,9.
Traditionally, tritium (3H) radio-labelled cholesterol has been used in cholesterol efflux10. The emission signal of radioisotopes is highly sensitive10; however, radio-labelled cholesterol presents obvious handicaps such as long protocols, risk of exposure to ionizing radiation, and the need for special radioactivity facilities and equipment to ensure safe handling of radioactive emission. On the contrary, fluorescence has been successfully incorporated in diagnostic techniques due to its simplicity in fluorescent signal detection, the wide variety of fluorophores available, and its safety11. Several fluorescent-labelled sterols have been used to study cholesterol metabolism including dehydroergosterol (with intrinsic fluorescence), dansyl cholesterol, 4,4-difluoro-3a,4adiaza-s-indacene (BODIPY)-cholesterol, and 22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3&#946;-Ol (NBD-cholesterol). Particularly, NBD-cholesterol presents an efficient uptake in human cells12. Two different NBD labelled-cholesterol are currently available: 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (22-NBD) and 25-(N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino)-27-norcholesterol (25-NBD; Figure 1). Cholesterol labelled with 22-NBD moiety may best suit cholesterol efflux studies, while 25-NBD-cholesterol is mainly used in cellular membrane dynamics research13,14.
Cell lines typically used in in vitro cholesterol efflux assays are monocyte-like cells such as human leukemia-derived THP-1 cells, murine Raw 264.7 cells15, or J774.1. All of these cells can be differentiated into macrophages in vitro using phorbol 12-myristate 13-acetate (PMA), but THP-1-derived macrophages (dmTHP-1) best reflect and mimic the human macrophages16.
In the present study, we optimize and standardize a fluorescent high-throughput method to determine the cholesterol efflux capacity of serum samples on dmTHP-1, using 22-NBD-cholesterol as an alternative to [3H]-cholesterol. In addition, we compare the optimized fluorescent technique with the standard radioactive analog.

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

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.

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

Watch this Scientific Journal Video about High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay at JoVE.com

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

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 ...

high-throughput-nitrobenzoxadiazole-labeled-cholesterol-efflux-assay

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8.9K Views.  Hospital Clínic de Barcelona. 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.

Video: High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

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 ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Biochemistry

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 ...

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 ...

Biology

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 ...