We would like to thank the FIOCRUZ (&#34;Programa de excel&#234;ncia em pesquisa b&#225;sica e aplicada em sa&#250;de dos laborat&#243;rios do Instituto Le&#244;nidas e Maria Deane - ILMD/Fiocruz Amaz&#244;nia-PROEP-LABS/ILMD FIOCRUZ AMAZ&#212;NIA&#34;), the Post-graduate Program in Biotechnology (PPGBIOTEC at the Universidade Federal do Amazonas - UFAM), the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel (CAPES), and the Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Amazonas (FAPEAM) for providing the scholarships. Figure 2&#160;and Figure 4&#160;were created with biorender.com.

NOTE: See the Table of Materials for details related to all the materials, reagents, and instruments used in this protocol.
1. Extraction of IgY from egg yolks
<ol>
	<li>Hygienization of the eggs
	<ol>
		<li>Immerse the eggs (freshly laid or until 4 days post-laid, from the Gallus gallus Dekalb White lineage) in a 0.2% diluted solution of sodium hypochlorite, rinse quickly under running water, and wipe gently for later or immediate use. 
		NOTE: If not used immediately, keep at 4 &#176;C for up to 15 days.</li>
	</ol>
	</li>
	<li>Separation of the yolks
	<ol>
		<li>Break the egg carefully, and separate the yolk from the white with the help of a yolk separator.</li>
		<li>Remove the excess white with the help of filter paper. Pierce the yolk, and collect its interior into a 50 mL conical tube. Store the yolk at &#8722;20 &#176;C for at least 24 h prior to use. 
		NOTE: During these experiments (data not shown), it was observed that the use of yolks that had not been frozen previously hindered the isolation of immunoglobulins from the lipid portion of the yolks.</li>
	</ol>
	</li>
	<li>Acidification of the yolks
	<ol>
		<li>Thaw the stored yolk and dilute it at a 1:10 ratio in 1 mM PBS. Adjust the pH of the solution to 5 with 1 N HCl, and incubate the solution at 4 &#176;C for 6-24 h.</li>
		<li>Centrifuge the solution at 3,000 &#215; g for 40 min, recover the supernatant, and filter it using a 0.7 mm cellulose filter.</li>
	</ol>
	</li>
	<li>Precipitation of lipids with caprylic acid
	<ol>
		<li>Readjust the pH of the supernatants to 5, and add the caprylic acid (&#8805;98% initial concentration) to a final concentration of 8.7% under constant stirring for 30 min at 4 &#176;C. 
		NOTE: The volume of the supernatant may vary depending on the lipid mass precipitated in step 1.3.2. Thus, for example, 3.107 mL of caprylic acid should be added to 35 mL of the supernatant obtained after filtration.</li>
		<li>Centrifuge the samples for 15 min at 18,600 &#215; g, and separate the supernatant from the precipitated material.</li>
		<li>Readjust the pH of the solution to 7.4 using 1 M NaOH, quantify the antibodies using the Bradford assay8, and store the antibodies at &#8722;20 &#176;C until the time of use. 
		NOTE: The antibodies were obtained at a concentration of 1 &#181;g/&#181;L, which was verified by means of a 12% SDS-PAGE gel.</li>
	</ol>
	</li>
</ol>
2. Saturation curve of IgY antibodies to latex beads
<ol>
	<li>Coupling of the beads to the IgY antibodies
	<ol>
		<li>Add 21 &#181;L of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, initial concentration of 367 mM) and 21 &#181;L of N-hydroxysuccinimide (NHS, initial concentration of 50 mM) to 21 &#181;L of latex beads (4% w/v), and adjust to a final volume of 2.1 mL with filtered 10 mM PBS. 
		NOTE: The pH range of the PBS solution should be between 7.2 and 7.4, since in this work, a negative change in the coupling and other steps was observed when using a solution outside this range.</li>
		<li>Incubate at 22 &#176;C with rapid shaking for 3 h.</li>
		<li>Add 4 &#181;g, 2 &#181;g, 1 &#181;g, 0.5 &#181;g, 0.25 &#181;g, 0.125 &#181;g, and 0 &#181;g of the previously extracted capture antibody IgY-PfHRP2 (see step 1.4.3) to different microtubes containing 100 &#181;L of the solution described in step 2.1.1 (0.04% final bead concentration), and incubate under the same conditions as described in step 2.1.2 for 16 h.</li>
		<li>Centrifuge the samples at 857 &#215; g at 15 &#176;C for 5 min, discard the supernatants, and wash the latex beads twice with 500 &#181;L of filtered 10 mM PBS (0.008% final bead concentration) using centrifugation and resuspension cycles. 
		NOTE: Do not exceed 857 &#215; g, as in this work, it was observed that beyond this speed the beads undergo deformations that negatively alter the test result.</li>
	</ol>
	</li>
	<li>Blocking the beads
	<ol>
		<li>Resuspend the samples in 1 mL of blocking buffer (0.004% final bead concentration) (filtered 10 mM PBS + bovine serum albumin, BSA, 5%), and incubate under the same conditions as described in step 2.1.2 for 2 h.</li>
		<li>After blocking, wash as described in step 2.1.4, and resuspend in 10 mM PBS buffer.</li>
	</ol>
	</li>
	<li>Incubation with anti-chicken secondary antibodies labeled with Alexa Fluor 488
	<ol>
		<li>Add 100 &#181;L of fluorescent anti-chicken antibody (2 mg/mL) diluted to 1:2,000 in 10 mM PBS + 0.5% BSA to each microtube, as described in step 2.1.3.</li>
		<li>Incubate the samples as described in step 2.1.2, this time in the dark, for 30 min.</li>
		<li>Wash the beads as in step 2.1.4, and resuspend with 250 &#181;L of (0.016% final bead concentration) filtered 10 mM PBS. Perform the flow cytometer reading as described in section 5. 
		NOTE: From the fluorescence percentage data obtained, observe the initial point of plateau formation (1 &#181;g, as observed in this study). We recommend using the plateau&#39;s second point (2 &#181;g, as observed here) for the next steps (Figure 1).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65123/65123fig01.jpg" /> 
Figure 1: Graph of the flow cytometry analysis to determine the saturation point of the IgY-PfHRP2 antibody coupling to the latex beads. The x-axis represents the antibody concentration, and the Y-axis represents the percentage fluorescence obtained. Kruskal-Wallis test, p &#60; 0.0033. <a href="https://www.jove.com/files/ftp_upload/65123/65123fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Flow cytometry assay based on latex beads for antigen detection
<ol>
	<li>After determining the saturation point of the IgY antibodies to the latex beads, perform the bead activation process again, according to step 2.1.1.
	<ol>
		<li>Couple the beads at the observed saturation concentration (as described in step 2.1.3), and incubate according to step 2.1.2. 
		NOTE: In the assays performed using the IgY-PfHRP2 antibody, the saturation condition was 2 &#181;g for every 1 &#181;L of latex beads.</li>
	</ol>
	</li>
	<li>Block and wash the beads according to step 2.2.</li>
	<li>Incubation with the antigen
	<ol>
		<li>To determine the limit of detection, distribute the equivalent of 100 &#181;L of the blocked bead preparation into tubes containing different amounts of the recombinant protein PfHRP2 (1 &#181;g/mL) (rPfHRP2, 10 &#181;g, 1 &#181;g, 0.1 &#181;g, 0.01 &#181;g, 0.001 &#181;g, 0.0001 &#181;g, and 0 &#181;g) diluted in 10 mM PBS + 0.5% BSA in triplicate. 
		NOTE: This recombinant protein was developed in the work of Sousa et al.9.</li>
		<li>Incubate the bead solutions with rPfHRP2 protein as described in step 2.1.2 for 1 h.</li>
		<li>Perform washings as described in step 2.1.4, and discard the supernatant after centrifugation.</li>
	</ol>
	</li>
	<li>Incubation with the primary antibody
	<ol>
		<li>Add 2 &#181;g of mouse IgG antibody against the rPfHRP2 protein diluted in 10 mM PBS + 0.5% BSA to a final volume of 100 &#181;L in each sample. Incubate the samples as described in step 2.1.2, and discard the supernatant after the last centrifugation. 
		NOTE: This antibody was developed in the work of Sousa et al.9.</li>
	</ol>
	</li>
	<li>Incubation with the secondary antibody
	<ol>
		<li>Add 100 &#181;L of fluorescent anti-mouse antibody (2 mg/mL) diluted to 1:2,000 in 10 mM PBS + 0.5% BSA to each sample. Incubate as described in step 2.1.2, and resuspend in 250 &#181;L (0.016% final bead concentration) of filtered 10 mM PBS. Perform the reading according to section 5.</li>
	</ol>
	</li>
</ol>
4. Flow cytometer reading of the samples
<ol>
	<li>Cytometry setting: Alignment of the optical parameters of the cytometer coupled to a computer
	<ol>
		<li>Turn on the computer and flow cytometer, and wait a few minutes for the equipment to connect to the computer.</li>
		<li>Click on the cytometry software installed on the computer and log in to it. From the software workspace, select Cytometer | Startup | Clean mode | SIT fluids. 
		NOTE: Air bubbles and obstructions should be removed during the cytometer initiation process prior to sample acquisition.</li>
	</ol>
	</li>
	<li>Quality control
	<ol>
		<li>Use the quality control reagent to check the voltages of the photomultiplier tubes and assess the cytometer&#39;s sensitivity.</li>
		<li>Place the cytometer setup reagent and tracking beads in a 12 mm x 75 mm capped polystyrene tube.</li>
		<li>Gently mix the tube by vortexing.</li>
	</ol>
	</li>
	<li>Preparation of the suspension beads
	<ol>
		<li>For defining the baseline, add 0.5 mL of diluent (10 mM filtered PBS, pH 7) and three drops of beads to the 12 mm x 75 mm capped polystyrene tube. Vortex the tube gently before use.</li>
		<li>In the cytometer software workspace, select Cytometer | CST, and wait for the cytometer to disconnect from the cytometer software interface and connect to the CS&#38;T interface. Observe the following message on the cytometer software Status Bar at the bottom of the screen: Disconnected Cytometer.</li>
		<li>Use the 12 mm x 75 mm capped polystyrene tube containing the quality control reagent and attach it to the flow cytometer probe.</li>
	</ol>
	</li>
	<li>Verification of the cytometer configuration
	<ol>
		<li>In the System Summary window, verify that the cytometer configuration is appropriate for the experiment.</li>
	</ol>
	</li>
	<li>Setup for a performance check
	<ol>
		<li>Select the setup beads&#39; ID corresponding to the lot of CS&#38;T research beads.</li>
	</ol>
	</li>
	<li>Performance check
	<ol>
		<li>Attach the tube to the flow cytometer probe, and click on the Setup Control window. Select Check Performance, and click on Run.</li>
		<li>Once the performance check is complete, wait for a dialog box to open. Do one of the following:
		<ol>
			<li>To view the Cytometer Performance report, click on View Report.</li>
			<li>To complete the performance check and return to the setup view of the workspace, click on Finish.</li>
			<li>Observe the final result of the morphometric and fluorescence sensitivity analysis that appears in System Summary | Cytometer Performance Result with the status PASSED.</li>
			<li>Remove the tube from the cytometer. Check the cytometer performance results displayed in the System Summary window.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Sample analysis using flow cytometry
<ol>
	<li>Log in to cytometer software.
	<ol>
		<li>From the cytometer software workspace, select Cytometer | Clean mode | SIT fluids.</li>
		<li>Adjust to the software workspace.</li>
		<li>Define the gating strategy based on the negative control particles&#39; morphometric and fluorescence characteristics (beads without fluorescence).</li>
		<li>To determine the cell morphometry, choose Dot Plot Plot Graphic for the analysis of FSC-A parameters using SSC-A (Supplemental Figure 1A).</li>
		<li>Determine single cells by SSC using Dot Plot Plot Graphic for the analysis of SSC-H in the y-axis and SSC-W in the x-axis (Supplemental Figure 1B), and determine single cells by FSC using Dot Plot Plot Graphic for the analysis of FSC-H in the y-axis and FSC-W in the x-axis (Supplemental Figure 1C).</li>
		<li>For the fluorescence analyses, choose FL1 Dot Plot Plots using FCS-A. Use a 12 mm x 75 mm stoppered polystyrene tube containing unlabeled samples and samples previously labeled with single-color Alexa Fluor 488 (Supplemental Figure 1D, E).</li>
	</ol>
	</li>
	<li>Acquire the sample in flow cytometry.
	<ol>
		<li>Use a 12 mm x 75 mm stoppered polystyrene tube containing the previously labeled samples. Mix the tube contents carefully, attach the tube to the flow cytometer probe, and left-click on Acquire.</li>
		<li>To set the power of the lasers, click on Parameters, and in the options below, adjust the voltage of the FSC to 182 V and the voltage of the SSC to 236 V. To set the threshold, click on Cytometer | Threshold, and in the options below, adjust the FSC to 500 V.</li>
		<li>Set the analysis gate to characterize particles by size and complexity, as well as to perform single-cell analysis.</li>
		<li>To remove autofluorescence, analyze the dye-free sample containing only the latex beads, and adjust the voltages of the detector: in the options below, adjust the voltage of the FL1 (FITC) to 332 V.</li>
		<li>Set the fluorescence analysis gate in quadrangular format from the negative point shown in the dot plot graph.</li>
		<li>After the morphometric and fluorescent analyses have been adjusted, configure the device for 50,000 events: under Acquisition, click on Record.</li>
	</ol>
	</li>
</ol>

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The authors declare no potential conflicts of interest with respect to the research, authorship, and publication of this article.

The method of precipitation of the IgY antibody by pH reduction followed by lipid separation using caprylic acid is efficient in isolating total antibodies without any loss in functionality. The method proposed here is simpler and cheaper than that reported by Redwan et al.11, which also used precipitation by acidification and caprylic acid but with a more complex and laborious protocol. This method also presents advantages over commonly used methodologies for IgY isolation from egg yolks, such as...

Among the immunoassay techniques aimed at diagnosing diseases, the cytometric bead assay has emerged as a highly sensitive and reliable approach, since it allows for the analysis of thousands of particles in a single assay1. This technique, in addition to having high productivity and allowing the use of smaller volumes of samples, also presents great flexibility, since it allows for the detection of several molecules, such as cytokines, adhesion molecules, antibody isotypes, and proteins2,3.
Different particles are used for the development of these assays, among them latex beads, which are an effective and low-cost input. These can present modifications on their surface, such as the presence of functional groups or proteins that allow the covalent or non-covalent coupling of certain molecules3,4,5.
These immunoassays use components such as antigens and antibodies to perform the detection of disease markers and commonly require antibodies from mammals such as mice, rabbits, and goats. This creates problems related to ethical issues, since the immunization of mammals generally requires many animals to obtain a good yield, as well as the frequent performance of procedures that lead to the suffering of the animals6,7. An alternative to this is the use of IgY antibodies isolated from the egg yolks of immunized chickens, since high concentrations of the specific antibodies against the inoculated antigens can be found in the yolks; the production of a chicken is equivalent to the production of 4.3 rabbits over the course of a year6,7.
Thus, the objective of this protocol is to provide a method for evaluating IgY antibodies obtained from chicken egg yolks using flow cytometry with latex beads. For this, we propose a standardization method for a cytometric bead immunoassay in sandwich format using latex beads. As a model, we used IgY antibodies directed to the Plasmodium falciparum histidine-rich protein II antigen (IgY-PfHRP2). We describe a method for extracting the antibodies, discuss the critical steps for defining the coupling concentration of these to the latex beads, and present an evaluation of the limit of detection of the antigen. The high accuracy of flow cytometry, coupled with the low cost of latex beads, make this technique applicable for the analysis of immunoassay tools, such as antibodies and antigens. This method can be used for the detection of diverse targets.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-Chicken IgY (H+L), highly cross-adsorbed, CF&#160;488A antibody produced in donkey</td>
			<td>Sigma-Aldrich</td>
			<td>SAB4600031</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG (H+L), F(ab&#8242;)2</td>
			<td>Sigma-Aldrich</td>
			<td>SAB4600388</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II</td>
			<td>BD Biosciences</td>
			<td>BF-FACSC2</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva CS&#38;T research beads (CS&#38;T research beads)</td>
			<td>BD Biosciences</td>
			<td>655050</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva software 7.0</td>
			<td>BD Biosciences</td>
			<td>655677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>Bio-Rad</td>
			<td>#5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Caprilic acid</td>
			<td>Sigma-Aldrich</td>
			<td>O3907</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5702 R</td>
			<td>Eppendorf</td>
			<td>Z606936</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloride 37% acid molecular grade</td>
			<td>NEON</td>
			<td>02618 NEON</td>
			<td></td>
		</tr>
		<tr>
			<td>CML latex, 4% w/v</td>
			<td>Invitrogen</td>
			<td>C37253</td>
			<td></td>
		</tr>
		<tr>
			<td>Megafuge 8R</td>
			<td>Thermo Scientific</td>
			<td>TS-HM8R</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide Hydrochloride Powder (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>E7750-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysuccinimide (NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>130672-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>1003335620</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Acs Cientifica</td>
			<td>P.10.0594.024.00.27</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>Acs Cientifica</td>
			<td>R09211000</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Mixer Heat/Cool</td>
			<td>KASVI</td>
			<td>K80-120R</td>
			<td></td>
		</tr>
	</tbody>
</table>

standardization of a cytometric bead assay based on egg-yolk antibodies

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

Figure 2&#160;provides a graphical representation of the extraction process of IgY antibodies via acidification, followed by separation using caprylic acid (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65123/65123fig02.jpg" /> 
Figure 2: Schematic representation of the extraction step of IgY antibodies and separation...

Watch this Scientific Journal Video about Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies at JoVE.com

Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has ...

standardization-cytometric-bead-assay-based-on-egg-yolk

Research

JoVE Journal

Biochemistry

710 Views.  Programa de Pós-Graduação em Biotecnologia da Universidade Federal do Amazonas - UFAM. This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Video: Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

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 ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...