Name: cell-free biochemical fluorometric enzymatic assay for high-throughput measurement of lipid peroxidation in high density lipoprotein
Uploaded: 2017-10-12T13:00:00.000+00:00
Duration: 7 min 29 s
Description: Watch this Scientific Journal Video about Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein at JoVE.com

The authors gratefully acknowledge the work of Dr Mohamad Navab, Alan Fogelman and Srinivasa Reddy for their key role in development of earlier iterations of this model. T.A.A. is supported by an RMIT University Vice-Chancellor&#39;s Postdoctoral Fellowship. AJ and AH are supported by NHMRC project grant 1108792. TK is supported by NIH grants NIH K08AI08272, NIH/NCATS Grant # &#181;L1TR000124.

All experiments using human biological samples were performed with ethics approval from the University of California Los Angeles, Los Angeles and the Alfred Hospital Human Ethics committee, Melbourne.
NOTE: There are many variations of the fluorochrome HDL function Assay (see discussion)32. Below we will describe the protocol that gives the most consistent and reproducible results. An overview of the assay is shown in Figure 2.
1. Specimen Processing
<ol>
	<li>Use fresh, non-hemolyzed serum (can be collected in serum separator tubes) or citrate plasma obtained after 12-14 hours fasting. If using cryopreserved samples, include appropriate control as described in this Protocol.</li>
</ol>
2. Day 0-Preparation for the assay
<ol>
	<li>Prepare working layout of experiment with the appropriate controls, samples and replicates. Run each sample at least in triplicates. A representative 96 well layout is shown in Figure 3.</li>
	<li>Calculate all volumes of reagents that will be used for the assay based on number of samples and replicates. 
	NOTE: Include an additional 10% volume in each working stock to make sure there is enough volume for each reagent for the specific experiment.</li>
	<li>Label all required tubes for all different steps of the assay e.g. for the separation of HDL, measurement of HDL-C (optional) and the fluorochromeassay.</li>
</ol>
3. Day 1-Preparation of controls
<ol>
	<li>Preparation of study-specific pooled control
	<ol>
		<li>Create a control sample from pooled plasma or serum of all study samples. If running 20 samples in each plate in triplicate, combine 10 &#181;L from each sample into a 200 &#181;L pooled control. Give a separate aliquot of this pooled control to the clinical laboratory to determine the HDL-C value of this control. 
		NOTE: Use this pooled control to standardize the assay and account for all possible known (e.g. type of matrix serum versus plasma, freeze-thaw cycles, cryopreservation) and unknown confounders that may affect experimental variability27,28.</li>
	</ol>
	</li>
	<li>Preparation of laboratory-specific quality control (QCs)
	<ol>
		<li>Prepare a large stock of HDL in each laboratory (for example HDL isolated from 5 mL of plasma from 10 healthy donors) and cryopreserve several aliquots of this stock to minimize freeze-thaw cycles.</li>
		<li>Use at least 10 different replicates from this stock to determine the average value of HDLox. An acceptable range of measured HDLox values for each QC sample included in each assay is within &#60;15% of the coefficient of variation of this average value.</li>
		<li>Give a separate aliquot of this pooled control to the clinical laboratory to determine the HDL-C value of this control (e.g. 40 mg/dL). 
		NOTE: A detailed approach how to use blood from blood banks to create these controls has previously been described 27,28,32. Use additional QCs as needed (e.g. one from a healthy donor known to have normal HDL function and one from a donor known to have largely abnormal HDL function.</li>
	</ol>
	</li>
	<li>Optimization of background signal and blank values (optional)
	<ol>
		<li>To minimize background, add the appropriate amount of catalase (1-4 U/mL) in the incubation medium to rapidly remove the formed H2O2 due to spontaneous air oxidation of the buffers. 
		NOTE: Since the values from the blank wells (no HDL) are being subtracted from the values of the HDL samples and the results are being reported relative to a pooled control (that is included in the same 96 well plate), we have found that this step does not practically affect the results. Thus, optimization of the background signal with catalase can be omitted, if needed.</li>
	</ol>
	</li>
</ol>
4. Day 1-Separation of HDL Cholesterol using HDL precipitation
NOTE: Use a commercially available standardized HDL Cholesterol precipitating reagent to isolate apoB depleted serum according to the manufacturer&#39;s instructions. These reagents are widely used in colorimetric assays to determine HDL cholesterol levels.
<ol>
	<li>Use fresh (or thaw) plasma or serum sample.</li>
	<li>Mix equal volumes (e.g. 80 &#181;L) of plasma and HDL Cholesterol Precipitating Reagent (20% w/v polyethylene glycol in glycine buffer at pH 10.0 (25 &#176;C)).</li>
	<li>Mix well by pipetting up and down.</li>
	<li>Centrifuge at 1000-2000 x g for 10 min.</li>
	<li>Aspirate the supernatant (HDL fraction).</li>
	<li>Ideally use immediately the isolated HDL for the fluorochromeassay of HDL lipid peroxidation. However, if several samples are run in the same day and other steps such as measurement of HDL-cholesterol concentration are also done, then store the isolated HDL at 4 &#176;C and use it the next day for the fluorochromeHDL function Assay.</li>
</ol>
5. Day 1-Determination of HDL-C in isolated HDL
NOTE: This is optional if the value of HDL-C from the clinical laboratory is used to normalize HDLox by HDL-C amount.
<ol>
	<li>Quantify the HDL-cholesterol from plasma, using standard colorimetric assays27. Add 50 &#181;L of Cholesterol Reagent in each well and determine cholesterol concentration using a colorimetric plate reader and a cholesterol standard provided in each kit.</li>
</ol>
6. Day 2-Preparation of reagents
<ol>
	<li>Prepare HRP and cholesterol solutions of HRP 5 U/mL solution (Range 1-10 U/mL).</li>
	<li>Prepare 20 mM fluorochrome (e.g., Amplex Red) solutions: Thaw a vial of fluorochrome reagent and DMSO to room temperature. Prior to use, dissolve fluorochrome reagent (1 mg) in 200 &#181;L of DMSO. Store stock solution frozen at &#8804;-20 &#176;C, protected from light.</li>
	<li>Prepare positive and negative controls: Use 1X &#39;reaction buffer&#39; without cholesterol and 20 mM H2O2 working solution as a negative and positive control, respectively.</li>
</ol>
7. Day 2-Fluorochrome Assay
<ol>
	<li>Add 50 &#181;L of 1x Reaction buffer as blank (negative control).</li>
	<li>Optional: Add 20 mM working solution of H2O2 as positive control in each plate.</li>
	<li>To minimize experimental variability and ensure that addition of reagents and samples are done consistently and in a timely manner, perform all additions of samples in a separate 96 well round-bottom, clear, polystyrene or polypropylene plate (Plate 1). Then use a multichannel pipette to transfer specific volumes into 3 96-well plates (Plates 2-4: polypropylene, flat bottom, black) (that have identical layout) (Figure 2, Figure 3).
	<ol>
		<li>For example, first add 160 &#181;L of isolated HDL into each well/sample of plate 1 and then with the use of multichannel pipette transfer 50 &#181;L of HDL-cholesterol from each well/sample into the 96-well black plates. Change tips between each row/column and use non-filtered tips for all the transfers.</li>
	</ol>
	</li>
	<li>Leave samples in the wells. Do not discard. There are no wash steps between addition of reagents.</li>
	<li>Add 50 &#181;L of HRP solution 5 U/mL (0.25 U) to each well. 
	NOTE: Use HRP before the addition of the fluorochrome reagent.</li>
	<li>Incubate for 30 min at 37 &#176;C. Do not discard samples after incubation (no wash steps between additions of reagents).</li>
	<li>Add 50 &#181;L of fluorochromereagent for a final concentration of 300 &#181;M. At this point, total reaction volume is 150 &#181;L. Mix well and protect from light.</li>
	<li>Assess the fluorescent readout (in the dark) every minute over 120 min at 37 &#176;C with a fluorescent plate reader (530/590 nm filters). 
	NOTE: Use a shorter 60-minute interval with fresh samples. We have found that 120-minute assay gives more reproducible data with the use of cryopreserved samples.</li>
	<li>Record data using appropriate software.</li>
</ol>
8. Day 3-Data analysis
<ol>
	<li>Record fluorescence units (arbitrary units) at 120 minutes after addition of the fluorochrome Reagent for all the samples including blank wells and all controls.</li>
	<li>Calculate the mean value of fluorescence units (based on at least triplicates) (HDLox_sample). Do not take outliers (&#62; 2 SDs from the average value) into consideration.</li>
	<li>Subtract the background fluorescence by using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/56325/56325eq1.jpg" /></li>
	<li>Normalization for HDL cholesterol amount (HDL-C): Normalize the HDLox lipid peroxidation value for each sample (including the pooled control) by the HDL-C) (mg/dl). Throughout the results section, HDLox is presented as n(HDL-C) HDLox measure to reflect the adjustment for HDL-C. Use the following equation: 
	<img alt="Equation 2" src="/files/ftp_upload/56325/56325eq2.jpg" /></li>
	<li>Standardize the expression of HDLox lipid peroxidation value of each sample against the value of a pooled control. Normalize the n(HDL-C) HDLox lipid peroxidation value for each sample (adjusted for HDL-C) by the n(HDL-C) HDLox lipid peroxidation value of the pooled control. Throughout the results section, HDLox is presented as nHDLox measure to reflect the adjustment for experimental variability and HDL-C. Use the following equation: 
	<img alt="Equation 3" src="/files/ftp_upload/56325/56325eq3.jpg" /> 
	NOTE: This approach is similar to other established experimental approaches to reduce experimental variability of measurements such as international normalized ratio (INR)44,45. This approach has been validated in clinical studies32,33,34,35,36,37,38,39.</li>
	<li>Perform quality control of experimental results using standard quality control samples. Each laboratory should establish their own quality controls (QCs). Ideally there is a QC for nHDLox from a sample with known dysfunctional HDL and a QC for nHDLox from a sample from healthy donors [e.g. use pooled sample from young adults who are established blood bank donors and have no known comorbidity and risk factors for cardiovascular disease including smoking]. The latter control standardizes results among different laboratories. The average values of these QCs are established based on at least 5 replicates (typically we use 10 replicates for this important step).
	<ol>
		<li>Check whether measured QCs in each assay have values within the expected range of values. Assuming an acceptable maximum experimental variability of 15%, all the measured experimental values should fall within 15% of the known average values of established QC. Use the following equations: 
		<img alt="Equation 4" src="/files/ftp_upload/56325/56325eq4.jpg" /> 
		<img alt="Equation 5" src="/files/ftp_upload/56325/56325eq5.jpg" /> 
		NOTE: If either of the included quality control samples (normal versus dysfunctional HDL) gives HDLox values outside expected range (established in each laboratory) then repeat the experiment.</li>
	</ol>
	</li>
	<li>Perform quality control of experimental results using the coefficient of variation (CV) to quantify experimental variability: Repeat all samples with CV% &#62; 15%. Use the following equation: 
	<img alt="Equation 6" src="/files/ftp_upload/56325/56325eq6.jpg" /> 
	NOTE: A typical intra-assay (within the same plate) and inter-assay (among different plates) experimental variability is &#60; 15%. 2) A typical intra-assay CV is between 1-7% and a typical interassay CV is between 3-10%.</li>
</ol>

This protocol and assay are relevant to the patent PCT/US2015/018147.

The protocol described here offers a robust tool to answer important research questions regarding the role of HDL function in atherosclerosis and human disease. The assay quantifies the HDL lipid peroxide content per mg of HDL-C using enzymatic amplification (HRP). This approach avoids known limitations of prior HDL function assays (e.g. the cholesterol efflux assay) including significant heterogeneity with regards to types of cells used, type of readout reported, lack of standardization and confounding effects of trigly...

Atherosclerotic cardiovascular disease (CVD) is the leading cause of death worldwide1,2. Epidemiological studies have shown that low levels of high-density lipoprotein (HDL) cholesterol are generally inversely associated with the risk for the development of atherosclerosis1,2. Although several studies support an atheroprotective role for HDL1,2, the mechanism by which HDL attenuates the initiation and progression of atherosclerosis is complex 3,4. Thus, it has been suggested that the complex structure and function of HDL rather than absolute level may more accurately predict atherosclerosis 5,6,7,8. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. These i) reduce its cholesterol efflux potential 9, ii) decrease anti-inflammatory and increase HDL-associated pro-inflammatory proteins 6,7, iii) decrease antioxidant factor levels and activity and HDLs ability to inhibit oxidation of Low Density Lipoprotein (LDLox)10 and iv) increase lipid hydroperoxide content and redox activity (HDLox)9,11. Robust assays that evaluate the pleotropic functions of HDL (such as cholesterol efflux, antioxidant function) may complement determination of HDL-HDL-C in the clinic.
HDL function is usually assessed by cell-based methods such as the cholesterol efflux assay8,12,13,14. These methods have major limitations including significant heterogeneity with regards to types of cells used, type of readout reported, lack of standardization and confounding effects of triglycerides 7,15. These drawbacks pose difficulties for large clinical studies16. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. The cholesterol efflux is one of the most important functions of HDL but it can only be determined by cell-based assays. Other approaches to determine HDL function such as proteomics17,18,19,20,21,22,23,24 and cell-based monocyte chemotaxis assays of HDL function 17,22,25 have not been standardized and cannot be used in large scale human studies.
HDL has significant antioxidant atheroprotective effect5,6,7,8. The antioxidant function of HDL has been determined in the presence of LDL in previous cell free fluorometric assays 26. These biochemical fluorometric methods of HDL antioxidant function were originally developed by Mohamad Navab and Alan Fogelman and their colleagues26. Although many human studies have used these methods to determine HDL function 17,18,19,20,21,22,23,24, lipid (HDL)-lipid (LDL) and lipid-fluorochrome interactions may limit reproducibility of these cell free non-enzymatic biochemical assays of HDL function27,28.
Recent interest has focused on the functional consequences of HDL oxidation that is the result of oxidation of both lipids and proteins within HDL 27,29,30. Prior studies have shown that oxidation of HDL impairs HDL function 27,29,30. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Thus HDL lipid peroxide content can be used to determine HDL function 9,17,20,31 and given the known limitations of prior assays of HDL function7,15,27,32, we developed an alternative fluorometric method that quantifies HDL lipid peroxide content (HDLox) 32. 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 32. The biochemical principle of the assay is shown in Figure 1. We have shown that this fluorescence-based approach does not have the limitations of prior HDL function assays27,28. This assay has been further refined and standardized in our laboratory so that it can reliably be used in large scale human studies even with cryopreserved plasma 32,33,34,35,36,37,38,39,40,41,42. 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 phenotypes32,33,34,35,36,37,38,39. Here, we describe this simple, yet robust method to measure HDL lipid peroxide content (HDLox). This assay can be used as a tool to answer important research questions regarding the role of HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis)32.

<table><tbody><tr><td>&lt;strong&gt;Experimental Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HDL PEG (Polyethylene Glycol) Precipitating Reagent</td><td>Pointe Scientific</td><td>H7511</td><td></td></tr><tr><td>Amplex Red reagent.</td><td>Life Technologies, Grand Island, NY</td><td>A12216</td><td>Amplex Red Cholesterol Assay Kit.&lt;br/&gt; &amp;bull; &amp;le;&amp;ndash;20&amp;deg;C &amp;bull; Desiccate&lt;br/&gt; &amp;bull; Protect from light</td></tr><tr><td>DMSO.</td><td>Life Technologies, Grand Island, NY</td><td>A12216</td><td>Amplex Red Cholesterol Assay Kit.&lt;br/&gt; &amp;bull; &amp;le;&amp;ndash;20&amp;deg;C &amp;bull; Desiccate&lt;br/&gt; &amp;bull; Protect from light</td></tr><tr><td>Horse Radish Peroxidase (HRP)</td><td>Life Technologies, Grand Island, NY</td><td>A12216</td><td>Amplex Red Cholesterol Assay Kit.&lt;br/&gt; &amp;bull; &amp;le;&amp;ndash;20&amp;deg;C &amp;bull; Desiccate&lt;br/&gt; &amp;bull; Protect from light</td></tr><tr><td>Cholesterol Esterase.</td><td>Life Technologies, Grand Island, NY</td><td>A12216</td><td>Amplex Red Cholesterol Assay Kit.&lt;br/&gt; &amp;bull; &amp;le;&amp;ndash;20&amp;deg;C &amp;bull; Desiccate&lt;br/&gt; &amp;bull; Protect from light</td></tr><tr><td>Cholesterol Reference standard</td><td>Life Technologies, Grand Island, NY</td><td>A12216</td><td>Amplex Red Cholesterol Assay Kit.&lt;br/&gt; &amp;bull; &amp;le;&amp;ndash;20&amp;deg;C &amp;bull; Desiccate&lt;br/&gt; &amp;bull; Protect from light</td></tr><tr><td>Resorufin fluorescense Reference standard</td><td>Life Technologies, Grand Island, NY</td><td>A12216</td><td>Amplex Red Cholesterol Assay Kit.&lt;br/&gt; &amp;bull; &amp;le;&amp;ndash;20&amp;deg;C &amp;bull; Desiccate&lt;br/&gt; &amp;bull; Protect from light</td></tr><tr><td>5x Reaction Buffer.</td><td>Life Technologies, Grand Island, NY</td><td>A12216</td><td>Amplex Red Cholesterol Assay Kit.&lt;br/&gt; &amp;bull; &amp;le;&amp;ndash;20&amp;deg;C &amp;bull; Desiccate&lt;br/&gt; &amp;bull; Protect from light</td></tr><tr><td>HDL Cholesterol Automated Reagent</td><td>ThermoFisher Scientific Co., San Jose, CA, USA.</td><td>TR39601</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasticware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well plates (polypropylene, flat bottom, clear).</td><td>Sigma Aldrich</td><td>M0687</td><td></td></tr><tr><td>96-well plates (polypropylene, flat bottom, black).</td><td>Sigma Aldrich</td><td>M9936</td><td></td></tr><tr><td>1.5 mL Eppendorf tubes</td><td>Eppendorf</td><td>0030 125.150</td><td></td></tr><tr><td>ClipTip 200, sterile</td><td>ThermoFisher Scientific Co., San Jose, CA, USA.</td><td>14-488-058</td><td></td></tr><tr><td>Thermo Scientific Multichannel Pipettes, 8-channel, 125&amp;nbsp;</td><td>ThermoFisher Scientific Co., San Jose, CA, USA.</td><td>&amp;nbsp;14-387--955</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gen5 2.01 software</td><td>Biotek, Vermont, USA</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gen5 Plate reader</td><td>Biotek, Vermont, USA</td><td>NA</td><td></td></tr></tbody></table>

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).

50 &#181;L of each HDL sample are added into each well as in step 7.3. 50 &#181;L of HRP solution 5 U/mL (0.25 U) are then added into each well as in step 7.5. Samples are incubated for 30 min at 37 &#176;C as in step 7.6. 50 &#181;L of fluorochrome reagent are then added into each well as in step 7.7 (final concentration of 300 &#181;M). The fluorescent readout (in dark) is then assessed every minute over 120 minutes at 37 &#176;C with a fluorescent plate reader (530/590 nm filters). Rep...

Watch this Scientific Journal Video about Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein at JoVE.com

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

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 ...

cell-free-biochemical-fluorometric-enzymatic-assay-for-high

Research

JoVE Journal

Immunology and Infection

9.2K Views.  University of California, Los Angeles. Hier beschrijven we een fluorimetrische cel-vrije biochemische bepaling voor bepaling van de HDL-lipide peroxidatie. Deze snelle en reproduceerbare kwantitatieve analyse kan worden gebruikt om te bepalen van HDL functie in grootschalige studies en kan bijdragen aan ons begrip van HDL functie in ziekten bij de mens.

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

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Biochemistry

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Chemistry

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

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 ...

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 ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Bioengineering

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 ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

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

Medicine

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 ...

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

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