a degranulation assay to monitor neutrophil activation and gelatinase release

A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

To begin, stimulate the neutrophils in RPMI1640, with or without 0.5 or 10 micromolar fMLP or 5 to 20 nanograms per milliliter TNF-alpha, and incubate for 10 minutes at 37 degrees Celsius.
At the end of the incubation, centrifuge the cells at 800 times g for 5 minutes. Collect the supernatant, and spin again, as demonstrated earlier.
To measure the specific granule degranulation, use the EnzChek gelatinase-collagenase assay kit. As per the manufacturer&#39;s instructions, add 1 milliliter of Milli-Q water to the lyophilized DQ gelatin vial to make 1 milligram per milliliter of DQ gelatin stock solution.
Then, prepare the reaction buffer by mixing 18 milliliters of Milli-Q water and 2 milliliters of 10X reaction buffer into a 15-milliliter tube. Add 0.5 milliliters of Milli-Q water to the Clostridium collagenase tube to prepare a 1,000 units per milliliter stock solution.
Before the experiment, dilute the collagenase standard solution using the reaction buffer to make different concentrations for generating the standard curve.
Next, add 80 microliters of the reaction buffer to each well of a 96-well plate. Dilute DQ gelatin stock solution four times using the reaction buffer before use. Then, add 20 microliters of diluted DQ gelatin solution to each well.
Add 100 microliters of sample or different concentrations of Clostridium collagenase standard solution into each well.
For data collection, insert the assay plate into the PHERAstar plate reader. Then, represent the data as a total activity using known concentrations of collagenase as a standard curve.

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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 neutrophil
<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.</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 PBS with 45 ml Percoll (Cytiva).</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 from 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 a break. Neutrophils are collected at the pellet.</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 RPMI1640 (Roswell Park Memorial Institute) media at 3 x 106 cells/ml and kept on ice before the experiment. Cells are used up 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% 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 gently underlay with 12 ml of 74% Percoll.</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 a break.</li>
		<li>Remove the top mononuclear cell layer and harvest the neutrophil layer (around 5 -8 ml) into a new 50 ml tube containing 40 ml HBSS.</li>
		<li>Centrifuge at 800 g for 10 minutes at 4 &#186;C to pellet neutrophils.</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.</li>
		<li>Centrifuge it at 800 g for 5 minutes at 4 &#186;C.</li>
		<li>Resuspend the cell at 3 x 106 neutrophils/ml concentration in RPMI1640 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-Formylmethionyl-leucyl-phenylalanine (fMLP) or 5 ng/ml tumor necrosis factor alpha (TNF-&#945;) for 10 minutes at 37&#176;C.</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 100 &#956;l supernatant was examined for degranulation assay.</li>
	</ol>
	</li>
	<li>Measurement of degranulation of gelatinase granules
	<ol>
		<li>The EnzChek Gelatinase/Collagenase Assay kit (Thermo Fisher Scientific) was used to measure the specific granule degranulation.</li>
		<li>According to the manufacturer&#39;s instructions, add 1 ml Milli-Q water to the lyophilized dye-quenched (DQTM) gelatin vial to make 1 mg/ml DQTM gelatin stock solution.</li>
		<li>Mix 18 ml Milli-Q water and 2 ml 10x reaction buffer into a 15 ml tube to make the reaction buffer.</li>
		<li>Add 0.5 ml Milli-Q water to the Clostridium collagenase tube to prepare a 1,000 U/ml stock solution. Before the experiment begins, dilute the collagenase standard solution using the reaction buffer to make the concentrations of collagenase 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 2 U/ml for generating a standard curve.</li>
		<li>In a 96-well plate, add 80 &#956;l of the reaction buffer to each well.</li>
		<li>Dilute DQTM gelatin solution 4 times from stock solution with 1x reaction buffer before use. Then, add 20 &#956;l of diluted DQTM gelatin solution to the wells of a 96-well plate to make the final concentration of DQTM gelatin 25 &#956;g/ml in the reaction.</li>
		<li>Add 100 &#956;l sample or different Clostridium collagenase standard solution concentrations into each well.</li>
		<li>Insert the assay plate into the PHERAstar plate reader (BMG Labtech) with an excitation wavelength of 485 nm and emission wavelength of 520 nm. The data point was collected every 10 minutes for 4 hours. Data were presented as a total activity using known concentrations of collagenase as a standard curve.</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>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</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</td>
			<td>315-01A</td>
			<td>Used for stimulating neutrophils</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</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 an assay to monitor neutrophil activation and granule exocytosis through a degranulation assay. Upon activation by proinflammatory compounds, cytoplasmic granules within neutrophils containing gelatinase fuse with the cell membrane, discharging their cargo. The presence of gelatinase in the supernatant is assessed through the hydrolysis of a fluorophore-conjugated gelatin substrate, confirming neutrophil activation and degranulation.

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

Neutrophils are immune cells containing cytoplasmic granules that store proteases, including gelatinase.
Take tubes containing neutrophils in a growth medium.
Add N-formyl-methionyl-leucyl- phenylalanine or fMLP &#8212; a proinflammatory synthetic bacterial peptide &#8212; to one tube. The other tube serves as a control. 
fMLP binds to its specific receptor on the neutrophil, triggering neutrophil activation.
The cytoplasmic granules migrate toward the plasma membrane. Membrane SNARE proteins mediate the granule and plasma membrane fusion, leading to cargo discharge and degranulation.
Centrifuge to pellet the cells. Transfer the gelatinase-containing supernatant to a multi-well plate containing a gelatinase substrate &#8212; a fluorophore-conjugated gelatin.
A high density of fluorophore labeling on the substrate causes intramolecular self-quenching of the fluorescence signal.
Gelatinase hydrolyzes the substrate, separating the fluorophore molecules and allowing fluorescence emission.
Using a microplate reader, measure fluorescence intensities in the wells to determine the concentration of released gelatinase following neutrophil activation and degranulation.

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In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

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Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

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A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

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