in vitro stimulation and visualization of macrophage extracellular traps

In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

Under sterile conditions, prepare the culture media containing different stimulators of MET release to the complete RPMI 1640 media. For experiments with hypochlorous acid stimulation, prepare 200 micromolar hypochlorous acid in a Falcon tube with HBSS that&#39;s been pre-warmed to 37 degrees Celsius.
After the polarization treatment, aspirate the cell media from each well, and carefully wash the cells three times with 1-milliliter aliquots of either sterile PBS for PMA, TNF-alpha, and interleukin-8 stimulation, or HBSS for hypochlorous acid stimulation.
After removing the PBS in the final washing step, add 1 milliliter of complete media containing PMA, TNF-alpha, or interleukin-8. Incubate the cells at 37 degrees Celsius and 5% carbon dioxide in a cell incubator for six hours for TNF-alpha stimulation, and 24 hours for PMA or interleukin-8 stimulation.
For experiments with hypochlorous acid, after removing the HBSS in the final washing step, add 1 milliliter of freshly prepared hypochlorous acid. Then, incubate the cells for 15 minutes at 37 degrees Celsius in the presence of 5% carbon dioxide in a cell incubator. After that, carefully aspirate the cell supernatant, and wash the cells three times with 1-milliliter aliquots of HBSS.
After removing the HBSS from the final wash step, add 1 milliliter of complete RPMI 1640 culture media. Then, incubate the cells for 24 hours at 37 degrees Celsius in the presence of 5% carbon dioxide in a cell incubator.
Prepare SYTOX green dye in HBSS at a concentration of 40 micromolar. Directly add 25 microliters of the dye to each well containing HMDM. Incubate cells at room temperature for 5 minutes in the dark. Then, place the HMDM in tissue culture wells on the microscope stage of an inverted fluorescent microscope for imaging.
Turn on a broad spectrum fluorescent light source, bright field light source, and inverted microscope installed with a high-resolution color digital camera. Rotate the filter wheel to the number 2 position for green fluorescence with excitation at 504 nanometers and emission at 523 nanometers.
Using the 5X objective, focus the image with the coarse focus, then the fine focus knobs on the microscope until the image appears sharp, clear, and focused. Switch the microscope to camera mode.
Start the associated software. Click the Play button to preview the image, and adjust the fine focus knob on the microscope until the image appears sharp, clear, and focused in the software preview window. Click the Capture button.
Within the software, click File, Save as the required image file type. On the microscope, rotate the filter wheel to the number 5 position for brightfield imaging, and repeat the capture procedures to obtain the corresponding brightfield image.

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JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

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1. Polarization of HMDM
<ol>
	<li>Under sterile conditions, prepare the M1 priming media by adding interferon &#947; (IFN&#947;; 20 ng/mL) and lipopolysaccharide (LPS; 1 &#956;g/mL) to the complete RPMI-1640 culture media. Prepare the M2 priming media by adding interleukin 4 (IL-4; 20 ng/mL) to the complete RPMI-1640 culture media.</li>
	<li>Under sterile conditions, aspirate media from the tissue culture plate wells that contain the Human monocyte-derived macrophages, HMDM, which have been seeded and cultured as described in section 1.</li>
	<li>Carefully wash the wells containing the cells 3x with sterile PBS (pre-warmed to 37 &#176;C), using 1 mL aliquots of PBS.</li>
	<li>Add 1 mL of either the M1 or M2 priming media to each well containing the HMDM (whichever is appropriate for the experiment).</li>
	<li>Incubate the cells for 48 h at 37 &#176;C in the presence of 5% CO2 in a cell incubator.</li>
</ol>
2. Stimulation of HMDM to induce MET Release
<ol>
	<li>Under sterile conditions, prepare the culture media containing different stimulators of macrophage extracellular traps MET release (whichever is appropriate for the experiment) to the complete RPMI-1640 media: PMA (25 nM), human recombinant TNF&#945; (25 ng/mL), or human recombinant IL-8 (50 ng/mL).</li>
	<li>For experiments with hypochlorous acid, HOCl stimulation, prepare HOCl (200 &#956;M) in HBSS (pre-warmed to 37 &#176;C), immediately before the addition to the cells. Ensure that the HOCl is not prepared in complete cell media. 
	NOTE: The concentration of the stock solution of HOCl is quantified by measuring the UV absorbance of the solution at 292 nm and pH = 116 and using an extinction coefficient of 350 M-1cm-1.</li>
	<li>After the polarization treatment described in section 2, aspirate the cell media from each well and carefully wash the cells 3x with 1 mL aliquots of either: sterile PBS (for PMA, TNF&#945; and IL-8) or HBSS (for HOCl), which have been pre-warmed to 37 &#176;C.</li>
	<li>For experiments with PMA, TNF&#945;, or IL-8: add 1 mL of the complete media containing PMA, TNF&#945;, or IL-8 after removing the PBS in the final washing step.</li>
	<li>For experiments with TNF&#945;, incubate the cells for 6 h at 37 &#176;C in the presence of 5% CO2 in a cell incubator. For experiments with PMA and IL-8, incubate the cells for 24 h at 37 &#176;C in the presence of 5% CO2 in a cell incubator.</li>
	<li>For experiments with HOCl, add 1 mL of HOCl in HBSS after removing the HBSS in the final washing step. Then, incubate the cells for 15 min at 37 &#176;C in the presence of 5% CO2 in a cell incubator.
	<ol>
		<li>Carefully aspirate the cell supernatant and wash the cells 3x with 1 mL aliquots of HBSS as described in step 2.3.</li>
		<li>After removing the HBSS from the final wash step, add 1 mL of complete RPMI-1640 culture media. Then, incubate the cells for 24 h at 37 &#176;C in the presence of 5% CO2 in a cell incubator.</li>
	</ol>
	</li>
</ol>
3. Visualization of MET in Live Cell Culture
<ol>
	<li>Prepare SYTOX green dye in HBSS at a concentration of 40 &#956;M.</li>
	<li>At the end of treatments described in section 2 to induce MET release, directly add 25 &#956;L of 40 &#956;M of the dye to each well containing HMDM.</li>
	<li>Incubate cells at room temperature (RT) for 5 minutes in the dark.</li>
	<li>Place the HMDM in tissue culture wells on the microscope stage of an inverted fluorescent microscope for imaging.</li>
	<li>Microscope procedures
	<ol>
		<li>Turn on a broad-spectrum fluorescent light source, brightfield light source, and inverted microscope installed with a high-resolution color digital camera (see Table of Materials).</li>
		<li>Rotate the filter wheel to the &#34;number 2&#34; position for green fluorescence (excitation = 504 nm, emission = 523 nm) for imaging of the green stained samples contained within the tissue culture wells.</li>
		<li>Using the 5x objective, focus the image with the coarse focus, then the fine focus knobs on the microscope, until the image appears sharp, clear, and focused when viewed through the microscope eyepiece.</li>
		<li>Switch the microscope to the camera mode.</li>
		<li>Start the associated software.</li>
		<li>Select the Capture tab on the software.</li>
		<li>Click the Play button to preview the image and adjust the fine focus knob on the microscope until the image appears sharp, clear, and focused in the software preview window.</li>
		<li>Click the Capture button. 
		NOTE: The captured image will automatically be displayed in the accompanying software.</li>
		<li>Within the software, click File | Save as the required image file type.</li>
		<li>On the microscope, rotate the filter wheel to the &#34;number 5&#34; position for brightfield imaging and repeat steps 3.5.2&#8211;3.5.9 to obtain the corresponding brightfield image.</li>
		<li>Repeat the steps 3.5.2&#8211;3.5.10 as necessary for subsequent image acquisition.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>120Q broad spectrum fluorescent light source</td>
			<td>EXFO Photonic Solutions, Toronto, Canada</td>
			<td>x-cite series</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning CellBIND Multiple Well Plate (12 wells)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3336</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Hypochlorous acid (HOCl)</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>Interferon gamma</td>
			<td>Thermo-Fisher</td>
			<td>PMC4031</td>
			<td>For M1 priming</td>
		</tr>
		<tr>
			<td>Interleukin 4</td>
			<td>Integrated Sciences</td>
			<td>rhil-4</td>
			<td>For M2 priming</td>
		</tr>
		<tr>
			<td>Interleukin 8</td>
			<td>Miltenyl Biotec</td>
			<td>130-093-943</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>59202C</td>
			<td>Added to culture media</td>
		</tr>
		<tr>
			<td>Olympus IX71 inverted microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12- myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>For washing steps</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>SYTOX green</td>
			<td>Life Technologies</td>
			<td>S7020</td>
			<td>For MET visulaization</td>
		</tr>
		<tr>
			<td>TH4-200 brightfield light source</td>
			<td>Olympus, Tokyo, Japan</td>
			<td></td>
			<td>x-cite series</td>
		</tr>
		<tr>
			<td>Tumor necrosis factor alpha</td>
			<td>Lonza</td>
			<td>300-01A-50</td>
			<td>For MET stimulation</td>
		</tr>
	</tbody>
</table>

Source: Zhang, Y., et al. <a href="https://app.jove.com/v/60541">In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages.</a> J. Vis. Exp. (2019).
This video demonstrates in vitro stimulation of macrophage extracellular traps (METs) in human monocyte-derive macrophages using different inflammatory stimuli such as TNF-alpha, PMA, and HOCL. The video also demonstrates the method of SYTOX dye staining of METs for visualization.

1. Polarization of HMDM

	Under sterile conditions, prepare the M1 priming media by adding interferon &#947; (IFN&#947;; 20 ng/mL) and lipopolysaccharide ...

Begin with an adherent macrophage culture.
Add pro-inflammatory cytokines.
The cytokine binds to the macrophage surface receptors, initiating a series of inflammatory events.
First, the cytokine signaling triggers the production of antimicrobial peptides.
Simultaneously, the chromatin within the macrophage nucleus undergoes decondensation.
This process, coupled with changes in membrane composition, disrupts the nuclear membrane, releasing decondensed chromatin into the cytoplasm.
In the cytoplasm, the antimicrobial peptide interacts with the released chromatin.
Cytokine signaling-mediated cell membrane permeabilization causes the decondensed chromatin to be released into the extracellular space, forming a Macrophage Extracellular Trap, or MET.
These web-like structures consist of DNA, histones, antimicrobial molecules, and enzymes.
Add SYTOX green, a fluorescent nucleic acid dye that selectively binds to the exposed DNA in the extracellular traps.
Under a fluorescence microscope, the MET appears as green streaks of extracellular DNA released from the stimulated macrophage.

47 Views. Stymulacja in vitro i wizualizacja pułapek zewnątrzkomórkowych makrofagów

Video: Stymulacja in vitro i wizualizacja pułapek zewnątrzkomórkowych makrofagów - Experiment

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

JoVE Journal

Immunology and Infection

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

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.

In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

Transkrypcja

In Vitro Stimulation and Visualization of Macrophage Extracellular Traps