an assay to monitor degranulation of azurophilic granules in neutrophils following activation

An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation

To begin, stimulate the mouse neutrophils in HBSS buffer containing 1 millimolar calcium chloride and magnesium chloride with or without fMLP or TNF-alpha for 10 minutes at 37 degrees Celsius.
Then, pellet the cells twice at 800 times g for 5 minutes. Collect 45 microliters of supernatant. Transfer the supernatant to the assay plate and run it in duplicates.
To detect the myeloperoxidase activity, add 80 microliters of 0.75 millimolar hydrogen peroxide solution, and 100 microliters of TMB solution to 20 microliters of supernatant. Then, incubate it at room temperature in the dark.&#160;
After 30 minutes, quench the reaction by adding 0.8 N hydrochloric acid. Read the absorbance on a PHERAstar plate reader at 450 nanometers. If necessary, generate a standard curve using recombinant myeloperoxidase to calculate the absolute amount of myeloperoxidase in the sample.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare mouse bone marrow neutrophils
<ol>
	<li>Harvest bone marrow cells
	<ol>
		<li>Bone marrow cells are flushed out from the femur and tibia of C57BL/6 mice with 10 ml Hank&#39;s balanced salt solution (HBSS) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.1% bovine serum albumin (BSA), pH 7.4 using a 10 ml syringe with 27 1/2G needle.&#160;</li>
		<li>Cells are filtered through 40 &#956;M nylon meshes and centrifuged at 300 g at 4&#176;C for 10 minutes.</li>
	</ol>
	</li>
	<li>Prepare 63% Percoll solution
	<ol>
		<li>Prepare 100% Percoll solution by mixing 5 ml sterile 10x phosphate-buffered saline (PBS) with 45 ml Percoll (Cytiva).&#160;</li>
		<li>Mix 3.15 ml 100% Percoll solution with 1.85 ml HBSS in a 15 ml tube to make a 63% Percoll solution.</li>
	</ol>
	</li>
	<li>Isolate neutrophils form bone marrow cells
	<ol>
		<li>Bone marrow cell pellets are resuspended in 1 ml HBSS; cells are laid carefully on the top of a 63% Percoll solution and centrifuged at 1,200 g at 4&#176;C for 30 minutes without break. Neutrophils are collected at the pellet.&#160;</li>
		<li>Red blood cells are lysed by a lysis buffer (Sigma), and cells are centrifuged at 800 g at 4&#176;C for 5 minutes.</li>
		<li>Neutrophils are resuspended with Roswell Park Memorial Institute 1640 (RPMI1640) media at 3 x 106 cells/ml and kept on ice before the experiment. Cells are used within 4 hours after isolation.</li>
	</ol>
	</li>
</ol>
2. Isolation of human neutrophils
<ol>
	<li>Collect leukocyte-rich layer from human blood
	<ol>
		<li>Collect blood into a syringe filled with 10% volume of sterile acid citrate dextran (ACD).</li>
		<li>Pour blood into a 50 ml tube and add the same volume of 3% Dextran T500 in 0.9% sodium chloride (NaCl).</li>
		<li>Let the mixture stay at room temperature for 10-15 minutes until red blood cells (the bottom layer) are separated from the leukocyte-rich layer (the top layer).</li>
		<li>Collect the top layer into a new tube.</li>
	</ol>
	</li>
	<li>Percoll gradient centrifuge
	<ol>
		<li>Prepare 55% and 74% Percoll solutions, as mentioned in Table 1.</li>
		<li>Prepare the Percoll gradient in 50 ml tubes by adding 12 ml of 55% Percoll and underlay with 12 ml of 74% Percoll very gently.</li>
		<li>Add the leukocyte-rich layer to the top of the Percoll gradient very gently.</li>
		<li>Spin cells at 800 g for 60 minutes at 12 &#186;C without break.</li>
		<li>Remove the top mononuclear cell layer and harvest the neutrophil layer &#160;&#160;&#160;&#160;&#160; (around 5 -8 ml) into a new 50 ml tube containing 40 ml HBSS.&#160;</li>
		<li>Centrifuge at 800 g for 10 minutes at 4 &#186;C to pellet neutrophils.&#160;</li>
		<li>Resuspend the cell pellet with 1 ml red blood cell (RBC) lysis buffer and incubate it for 2 minutes at room temperature.</li>
		<li>Transfer the cell suspension into a 1.5 ml tube and add 500 &#181;l HBSS.&#160;</li>
		<li>Centrifuge it at 800 g for 5 minutes at 4 &#186;C.</li>
		<li>Resuspend the cell at a concentration of 3 x 106 neutrophils/ml in RPMI1640 &#160;&#160;&#160;&#160;&#160;&#160;&#160; media containing 0.5% BSA. Store isolated neutrophils on ice.</li>
	</ol>
	</li>
</ol>
3. Specific granule degranulation assay
<ol>
	<li>Neutrophil stimulation
	<ol>
		<li>Neutrophils (2 &#215; 106/100 &#181;l, 200 &#956;l) in RPMI1640 were stimulated with or without 0.5 or 10 &#956;M N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) or 5 ng/ml tumor necrosis factor alpha (TNF-&#945;) for 10 minutes at 37&#176;C.&#160;</li>
		<li>Cells were then centrifuged at 800 g for 5 minutes.</li>
		<li>The supernatant was collected and spun for another 5 minutes at 800 g.</li>
		<li>The final supernatant, 100 &#956;l, was examined for degranulation assay.</li>
	</ol>
	</li>
	<li>Measurement of degranulation of azurophilic granules
	<ol>
		<li>Mouse neutrophils (2 &#215; 107 in 50 &#181;l) in HBSS buffer containing 1 mM calcium chloride (CaCl2) and 1 mM magnesium chloride (MgCl2) were stimulated with or without 0.5 or 10 &#181;M fMLP or 5 ng/ml TNF-&#945; for 10 minutes at 37&#176;C.&#160;</li>
		<li>Cells were spun at 800 g for 5 minutes twice to collect 45 &#956;l supernatant.</li>
		<li>The supernatant was run as duplicate samples.</li>
		<li>Incubate 20 &#956;l supernatant with 80 &#956;l 0.75 mM hydrogen peroxide (H2O2) solution and 100 &#956;l 3,3&#39;,5,5&#39;-tetramethylbenzidine Ready-to-Use solution at room temperature in the dark to detect the activity of myeloperoxidase (MPO).&#160;</li>
		<li>After 30 minutes, the reaction was quenched with the addition of 0.8 N hydrochloric acid (HCl), and the absorbance was read on a PHERAstar plate reader at 450 nm.</li>
		<li>If necessary, recombinant MPO can be used to generate a standard curve to calculate the absolute amount of MPO in the sample.</li>
	</ol>
	</li>
</ol>
Table 1. Component of Percoll gradient solution
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>55% Percoll (30 ml)</td>
			<td>74% Percoll (30 ml)</td>
		</tr>
		<tr>
			<td>Percoll (ml)</td>
			<td>16.5</td>
			<td>22.2</td>
		</tr>
		<tr>
			<td>9% NaCl (ml)</td>
			<td>1.83</td>
			<td>2.47</td>
		</tr>
		<tr>
			<td>0.9% NaCl (ml)</td>
			<td>11.67</td>
			<td>5.33</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EnzChek Gelatinase/Collagenase Assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>E12055</td>
			<td>Used for specific granule degranulation assay</td>
		</tr>
		<tr>
			<td>3,3&#39;,5,5&#39;-tetramethylbenzidine Ready-to-Use solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>J61325.EQE</td>
			<td>Used for azurophilic granule degranulation assay</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Millipore Sigma</td>
			<td>H1009</td>
			<td>Used for azurophilic granule degranulation assay</td>
		</tr>
		<tr>
			<td>Gibco&#8482; RPMI1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>21875034</td>
			<td>Used for culturing neutrophils</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A3294</td>
			<td>Used for culturing neutrophils&#160;</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Cytiva</td>
			<td>17089101</td>
			<td>Used for isolating neutrophils</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14170112</td>
			<td>Used for isolating and stimulating neutrophils</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>Used for isolating neutrophils</td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>70013032</td>
			<td>Used for isolating neutrophils</td>
		</tr>
		<tr>
			<td>Recombinant murine TNF-&#945;</td>
			<td>Peprotech&#160;</td>
			<td>315-01A</td>
			<td>Used for stimulating neutrophils&#160;</td>
		</tr>
		<tr>
			<td>fMLP</td>
			<td>Sigma</td>
			<td>F3506</td>
			<td>Used for stimulating neutrophils</td>
		</tr>
		<tr>
			<td>PHERAstar FSX plate reader&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate-dextrose solution (ACD)</td>
			<td>Sigma</td>
			<td>C3821</td>
			<td>Used for isolating human neutrophils</td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Pharmacosmos</td>
			<td>5510 0500 9007</td>
			<td>Used for isolating human neutrophils</td>
		</tr>
	</tbody>
</table>

This video demonstrates a degranulation assay of azurophilic granules in mouse neutrophils, involving the stimulation of neutrophils to release myeloperoxidase-containing azurophilic granules. The subsequent enzymatic reaction is employed to quantify myeloperoxidase levels, providing insight into neutrophil activation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare mouse bone marrow ...

Take tubes with mouse neutrophils suspended in a buffer.
These neutrophils contain intracellular granules, including primary, or azurophilic granules with myeloperoxidases.
Add N-formyl-methionyl-leucyl-phenylalanine or fMLP &#8212; a synthetic peptide, to one tube; the other serves as a control. Incubate.
fMLP binds to its specific receptor, triggering intracellular signaling, activating neutrophils, and increasing the cytoplasmic calcium concentration.
Further, the granules, including primary granules, translocate and fuse with the plasma membrane, mediated by calcium ions and membrane-bound proteins, resulting in increased degranulation into the buffer.
Post-incubation, centrifuge the tubes. Transfer the supernatant containing released cargo, including myeloperoxidase, into an assay plate.
Add hydrogen peroxide and tetramethylbenzidine. Incubate.
Myeloperoxidase utilizes hydrogen peroxide to oxidize tetramethylbenzidine, generating a blue-colored product.
Add an acidic solution to stop the reaction, changing the color to yellow.
Using a microplate reader, measure the product&#39;s absorbance, determining the concentration of secreted myeloperoxidase following neutrophil activation and degranulation.

121 조회수. An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation

비디오: An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation - 실험

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

미생물 및 화학적 자극에 의해 유도 된 호중구 세포 외 트랩의 형태 학적 및 조성 분석

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

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JoVE Journal

면역학 및 감염병학

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

변형된 샌드위치 ELISA를 사용하여 호중구 세포외 트랩에서 Myeloperoxidase-DNA 및 Neutrophil Elastase-DNA 복합체 정량화

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

To measure beta-hexosaminidase release, transfer 100 microliters of the cell suspension per well to a 96-well V-bottom well plate. Add 25 microliters of either stimulation or control solution to each well. Then, incubate the cells for 45 minutes at 37 degrees Celsius. Afterward, stop the reaction by placing the 96-well plate on ice for 5 minutes. Then, centrifuge the plate at 4 degrees Celsius for 4 minutes at 120 g.
Transfer 120 microliters of the supernatant to a flat-bottom 96-well plate, and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant. Next, add 125 microliters of lysis buffer to the cell pellets. Incubate the cell pellets for 5 minutes at room temperature, and resuspend them after the incubation by repeated pipetting.
Pipette 25 microliters of the 4-millimolar pNAG solution in the required wells of a new flat-bottom 96-well plate. Add 25 microliters of each supernatant, and 25 microliters of each cell lysate separately to the prepared pNAG solution. Incubate the reaction batches for 1 hour at 37 degrees Celsius. After the incubation, pipette 150 microliters of stop solution to each well to stop the reaction.
Next, analyze the plate sample light absorbance with a microplate reader at 405 nanometers with reference 630 nanometers for automatic background subtraction. In the acquisition program, check the box, &#34;Reference&#34;, and in the &#34;Absorbance&#34; program element menu, select &#34;630 nanometers&#34; for automatic background subtraction.

An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation

내레이션 대본

An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation