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><tbody><tr><td>Anti-Chicken IgY (H+L), highly cross-adsorbed, CF&amp;nbsp;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&amp;prime;)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&amp;amp;T research beads (CS&amp;amp;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&amp;prime;-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...

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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 ...

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Research

JoVE Journal

Biochemistry

778 Views.  Programa de Pós-Graduação em Biotecnologia da Universidade Federal do Amazonas - UFAM. Onder de immunoassay-technieken gericht op het diagnosticeren van ziekten, is de cytometrische kralentest naar voren gekomen als een zeer gevoelige en betrouwbare benadering, waarbij duizenden deeltjes in één enkele test worden geanalyseerd. De hoge nauwkeurigheid van flowcytometrie, in combinatie met de lage kosten van latexparels, maken onze techniek toepasbaar voor de analyse van immunoassays-instrumenten, zoals antilichamen en antigenen. IgY-antilichamen hebben lage kosten en de ethisch...

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

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Developmental Biology

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Immunology and Infection

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...

Multianalyte Immunobead Assay: A Flow-Based Immunoassay Technique for Concurrent Detection of Multiple Antibody Subtypes in Human Serum Sample

Source: Fhied, C. L. et al., <a href="https://www.jove.com/v/63352">Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19.</a> J. Vis. Exp. (2022).
This video describes an immunoassay technique using fluorescent magnetic beads or microspheres coupled with different antigens to simultaneously detect various antibody isotypes in a serum sample. The assay is helpful in understanding seroconversion during an infection or post-vaccination.

Source: Fhied, C. L. et al., Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19. J. Vis. Exp. (2022).
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Immunoassays

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

Source: Lehmann, J. S., et al. <a href="https://app.jove.com/v/56440">Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.</a> J. Vis. Exp. (2017).
This video demonstrates a multiplex bead-based immunoassay for quantifying multiple cytokine targets in a sample. A mixture of distinct microspheres conjugated to cytokine-specific antibodies is used to bind the cytokines present in the sample. Streptavidin-tagged reporters are immobilized on the bound-cytokines using biotinylated detection antibodies. Flow cytometry identifies the bound cytokines based on the unique sizes and internal fluorescence intensities of the microspheres, while the fluorescence intensity of the reporters indicates the number of cytokines.

1. Biological Sample Preparation
NOTE: The chosen anatomical site and volume of blood collection are left to the discretion of each individual ...

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

Source: Baxter, R. M., et al. <a href="https://app.jove.com/v/57780">Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry.</a> J. Vis. Exp. (2018).
This video highlights a single-cell proteomic method that employs heavy-metal-conjugated antibodies for distinct cell barcoding. Subsequently, immunostaining and mass cytometry are applied to evaluate immune phenotypic and functional alterations in human whole blood-derived immune cells.

1. Barcode of Lysed/Fixed Blood Cells

	Thaw the lysed/fixed cell samples from the -80 &#176;C storage slowly on ice; a maximum of 20 samples can be ...

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

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