Name: Video: Analiza aktywacji bazofili w obecności testowego alergenu za pomocą cytometrii przepływowej - Concept
Uploaded: 2023-11-30T12:47:35.000+00:00
Duration: 1 min 42 s
Description: 138 Views. Źródło: Doña, I. et al., Test aktywacji bazofili do diagnozy alergii. J. Vis. Exp. (2021) W tym filmie pokazujemy test do oceny aktywacji bazofili w obecności testowego alergenu za pomocą cytometrii przepływowej.

eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sample preparation
<ol>
	<li>Collect peripheral blood in 9 mL heparinized tubes and maintain the sample at room temperature (RT) in a rotor until it is required for the experimental protocol.</li>
	<li>Label 5 mL cytometer tubes for negative controls (2 tubes), positive controls (2 tubes), and different concentrations of allergen/drug (1 tube per each allergen/drug concentration tested). Place the tubes in a rack where tubes fit perfectly without slipping.</li>
	<li>Prepare stimulation buffer in double distilled water containing 2% (v/v) HEPES, 78 mg/L NaCl, 3.7 mg/L KCl, 7.8 mg/L CaCl2, 3.3 mg/L MgCl2, 1 g/L HSA. Adjust pH to 7.4 and add IL-3 at 2 ng/mL. Usually, prepare 100 mL and divide into 2.5 mL aliquots to be frozen at -20 &#176;C.</li>
	<li>Prepare positive controls in PBS-Tween-20 0.05% (v/v) (PBS-T): Positive control 1, N-Formyl methionyl-leucyl-phenylalanine (fMLP) (4 &#181;M), to confirm the quality of basophils; Positive control 2, Anti-IgE (0.05 mg/mL) as an IgE-mediated positive control.</li>
	<li>Prepare the allergen/drug in PBS-T at 2x the desired final concentration. 
	NOTE: Optimal allergen/drug concentrations to be used must be previously determined by using a wide range of concentrations, by dose-response curves, and by cytotoxicity studies following the same protocol steps.</li>
</ol>
2. Staining mix preparation
<ol>
	<li>Add monoclonal antibodies labeled with fluorochrome to the stimulation buffer following the manufacturer&#39;s recommended antibody concentration or by previous antibody titration. In this protocol, we add 1 &#181;L of each antibody (CCR3-APC and CD203c-PE for basophil identification; CD63-FITC for basophil activation) per 20 &#181;L of stimulation buffer. 
	NOTE: Protect the staining mix preparation from the light.</li>
	<li>Add 23 &#181;L of staining mix to each tube.</li>
</ol>
3. Blood stimulation
<ol>
	<li>Add 100 &#181;L of PBS-T to tubes 1 and 2 (negative control), 100 &#181;L of fMLP to tube 3, 100 &#181;L of anti-IgE to tube 4, and 100 &#181;L of the different allergen/drug concentrations to the following tubes. Incubate for 10 min at 37 &#176;C in a thermostatic bath with medium agitation to prewarm reagents.</li>
	<li>Gently add 100 &#181;L of blood to each tube to avoid hemolysis. Gently vortex tubes and incubate for 25 min at 37 &#176;C in a thermostatic bath with medium agitation.</li>
	<li>Stop the degranulation, keeping tubes at 4 &#176;C for at least 5 min. 
	NOTE: The protocol can be paused here at 4 &#176;C for 30-45 min if required.</li>
</ol>
4. Erythrocytes lysing
<ol>
	<li>Add 2 mL of 1x lysing buffer to each tube to lyse erythrocytes. Vortex each tube and incubate for 5 min at RT. 
	NOTE: In this step, cells are fixed due to fixative agents (formaldehyde) contained in the buffer.</li>
	<li>Centrifuge at 300 x g at 4 &#176;C for 5 min. Decant the supernatant, overturning the rack into a sink. Cells remain at the bottom of the tubes.</li>
	<li>Add 3 mL of PBS-T to each tube to wash cells. Vortex each tube.</li>
	<li>Centrifuge at 300 x g at 4 &#176;C for 5 min. Decant the supernatant, overturning the rack into a sink. 
	NOTE: Keep samples at 4 &#176;C, protected from the light until flow cytometer acquisition.</li>
</ol>
5. Flow cytometry acquisition
<ol>
	<li>Acquire samples by flow cytometer (e.g., BD FACSCalibur Flow Cytometer). Connect the flow cytometer to the computer software and wait for the cytometer to be ready. Load the template and instrument settings (Table 1).</li>
	<li>Start sample acquisition.</li>
	<li>Use the following cytometer strategies for the selection of activated basophils.
	<ol>
		<li>Gate the lymphocytes from the Side Scatter (SSC) - Forward Scatter (FSC) plot.</li>
		<li>Gate the basophils from the lymphocyte population as CCR3+CD203c+ cells. Acquire at least 500 basophils per tube.</li>
		<li>Show a CCR3 - CD63 plot to analyze activation using CD63 as an activation marker. Set the CD63 negative threshold to approximately 2.5% using the negative control tubes.</li>
		<li>Acquire all the samples.</li>
	</ol>
	</li>
</ol>
Table 1: Flow cytometer requirements
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Light Sources (lasers)</td>
			<td>488 nm Coherent SapphireTM air-cooled argon-ion laser; 20 mW; 633-nm JDS UniphaseTM HeNe air-cooled laser; 17 mW</td>
		</tr>
		<tr>
			<td>Light excitatory wavelength</td>
			<td>Blue laser:488 nm; Red laser: 633 nm</td>
		</tr>
		<tr>
			<td>Light source power at the excitatory wavelength</td>
			<td>Blue laser: 20 mW; Red laser: 17 mW</td>
		</tr>
		<tr>
			<td>Optical filters</td>
			<td>SSC: 488/10; FITC: 530/30, PE:585/42, APC: 660/20</td>
		</tr>
		<tr>
			<td>Optical detectors</td>
			<td>FSC, SSC, FL1-H FITC, FL2-H PE, FL4-H APC</td>
		</tr>
		<tr>
			<td>Optical detectors tye</td>
			<td>Air-cooled argon-ion laser</td>
		</tr>
		<tr>
			<td>Optical paths</td>
			<td>BD octagon (488 nm laser line); BD Trigons (633 nm laser line)</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube, without Cap, Nonsterile</td>
			<td>Corning</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-human CD193 (CCR3) Antibody</td>
			<td>BioLegend</td>
			<td>310708</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCalibur Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD63 Antibody</td>
			<td>BioLegend</td>
			<td>353006</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Thermo-Fisher</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Solution 10x concentrated</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD203c (E-NPP3) Antibody</td>
			<td>BioLegend</td>
			<td>324606</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Mouse Anti-Human IgE</td>
			<td>BD Biosciences</td>
			<td>555857</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-3</td>
			<td>R&#38;D Systems</td>
			<td>203-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheath Fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>TUBE 9 mL LH Lithium Heparin</td>
			<td>Greiner Bio-One</td>
			<td>455084</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing basophil activation in the presence of a test allergen using flow cytometry

Source: Do&#241;a, I. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62600">Basophil Activation Test for Allergy Diagnosis.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the test to evaluate basophil activation in the presence of a test allergen using flow cytometry.

Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sample preparation

	Collect ...

Take cytometer tubes containing a staining mixture comprising fluorescently labeled monoclonal antibodies suspended in a stimulation buffer.
Add decreasing concentrations of the test allergen into several tubes. Add anti-IgE antibodies to one tube to serve as the IgE-mediated positive control, and add buffer to another tube for the negative control.
Pipette human peripheral blood into the tubes. Incubate.
If the individual is sensitized to the test allergen, this exposure causes the allergen to bind to the allergen-specific IgE antibodies bound to Fc-receptors on the basophil surface.
Allergens crosslink IgE-bound Fc-receptors, causing basophil activation.
Basophils undergo degranulation, releasing intracellular granules containing inflammatory mediators, with the upregulation of CD63 and CD203c molecules.
Fluorescently-labeled monoclonal antibodies bind to CD63, CD203c, and CCR3 on basophils, respectively.
Lower the temperature to stop basophil degranulation.
Add buffer to lyse erythrocytes and fix the cells.
Using a flow cytometer, identify the basophils from the lymphocyte population.
Using CD63 as an activation marker, analyze basophil activation at decreasing allergen concentrations.

analyzing-basophil-activation-presence-test-allergen-using-flow

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

138 Views. Źródło: Doña, I. et al., Test aktywacji bazofili do diagnozy alergii. J. Vis. Exp. (2021) W tym filmie pokazujemy test do oceny aktywacji bazofili w obecności testowego alergenu za pomocą cytometrii przepływowej. 

Video: Analiza aktywacji bazofili w obecności testowego alergenu za pomocą cytometrii przepływowej - Concept

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

JoVE Journal

Immunology and Infection

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

Medicine

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry

Transkrypcja

Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry