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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. NETs Generation
<ol>
	<li>Stimulate neutrophils with 500 nM of PMA (per 30 ml of neutrophil solution) and incubate on a 150 x 25mm flat tissue culture dish with a 20 mm grid for 4 hr at 37 &#176;C 5% CO2. This will allow NETosis.</li>
	<li>After 4 hr of stimulation, gently aspirate and discard the media, leaving the layer of NETs and neutrophils adhered at the bottom. Do not disrupt this layer.</li>
	<li>Using a total of 15 ml of cold PBS without Ca and Mg per dish, wash the bottom of each dish by pipetting 15 ml of PBS on the bottom of the dish in order to lift off all adherent material from the bottom.</li>
	<li>Collect solution obtained from washing each dish (step 1.3) in a 15 ml conical tube and centrifuge for 10 min at 450 x g at 4 &#176;C. Neutrophils and any remaining cells will pellet at the bottom, leaving a cell-free NET-rich supernatant.</li>
	<li>Divide supernatant into 1.5 ml micro-centrifuge tubes and spin for 10 min at 18,000 x g at 4 &#176;C. This will allow all DNA to pellet. 
	NOTE: Centrifugation in larger tubes is easier and less time-consuming if such high speeds are attainable on the regular centrifuge, otherwise will need to divide supernatants in smaller tubes in order to use high-speed micro-centrifuge.</li>
	<li>Discard the supernatant and resuspend all pellets obtained together in ice-cold PBS to a concentration corresponding to 2 x 107 neutrophils per 100 &#956;l of PBS. This will yield the cell-free NET stock that can be used for subsequent experiments.</li>
	<li>Measure DNA concentration in the sample obtained using spectrophotometry or an alternate DNA quantification tool. An adequate concentration in the sample should range between 140 - 180 ng/&#956;l.</li>
</ol>

<table><tbody><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride</td><td>Wisent</td><td>311-425-CL</td><td></td></tr><tr><td>RPMI-1640 Medium with L-glutamine</td><td>Wisent</td><td>350-000-CL</td><td>3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum</td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma-Alrdrich</td><td>P8139</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>080-150</td><td></td></tr><tr><td>Integrid tissue culture dish with 20mm grid (150 x 25mm)</td><td>Falcon</td><td>353025</td><td></td></tr></tbody></table>

an in vitro technique to generate human neutrophil extracellular traps

Source: Najmeh, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52687">Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cytoplasmic proteins.

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

Source: Najmeh, S., et al. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (2015)
This video demonstrates an ...

Neutrophil extracellular traps, or NETs, are web-like structures of chromatin fibers complexed with antimicrobial proteins that trap and neutralize pathogens. NETs are secreted by neutrophils &#8212; immune cells that store antimicrobial proteins in specialized cytoplasmic granules.
To study NET formation in vitro, take a suspension of isolated neutrophils. Add phorbol myristate acetate, or PMA &#8212; a lipophilic pro-inflammatory compound. Plate the cells, and incubate.
PMA crosses the neutrophil membrane and enters the cytoplasm, where it binds to and activates protein kinase C or PKC.
Activated PKC induces downstream signaling to phosphorylate cytoplasmic components of NADPH oxidase &#8212; an enzyme complex &#8212; causing its assembly on the plasma and granule membrane. The complex produces reactive oxygen species or ROS.
The ROS cause the release of proteases and antimicrobial proteins from the granules. Granular proteases, along with ROS-activated citrullination enzymes, translocate to the nucleus.
The granular proteases cause partial histone degradation, while the citrullination enzymes promote histone citrullination &#8212; neutralizing positive charges. These modifications disrupt the histone-DNA interactions, resulting in chromatin decondensation.
The modification of the nuclear lamina causes nuclear envelope rupture and chromatin externalization; the chromatin mixes with the cytoplasmic antimicrobial proteins, forming NETs. The permeabilization of the cell membrane causes the extracellular release of NETs.
Discard the medium without disturbing the NETs and neutrophils adhered at the bottom. Resuspend the adhered components in a buffer.
Centrifuge to precipitate the cells &#8212; leaving a NET-rich supernatant ready for downstream assays.

an-in-vitro-technique-to-generate-human-neutrophil-extracellular-traps

Universidad de Cartagena UDEC

Research

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Immunology

Immune Systems and Components

Cell Differentiation Techniques

209 Views. Source: Najmeh, S., et al. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (2015) This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cy...

Video: An In Vitro Technique to Generate Human Neutrophil Extracellular Traps - Concept

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

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 ...

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

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

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

Transcript

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

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