Name: Time-lapse Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Analyze Post-adhesion Interaction between Cancer Cells and NETs
Uploaded: 2023-04-30T13:31:55
Description: Source: Kanamaru, R. et al. Neutrophil Extracellular Traps Generated by Low-density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell ...

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1.&#160;Time Lapse Video Analysis of the Trapped Tumor Cells
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	<li>Culture the peritoneal LDNs in a 35 mm round dish coated with poly-L-lysine and incubate for 2 h at 37 &#730;C in 5% CO2.</li>
	<li>Add the green-fluorescent dye for staining the nucleus and chromosomes to visualize NETs at the final concentration of 5 &#181;M.</li>
	<li>After 1 min of incubation, remove the media and add 2 mL of 0.1% BSA + RPMI 1640.</li>
	<li>Remove the media and add unstained 1 x 106&#160;MK...

time-lapse fluorescence microscopy of neutrophil extracellular traps or nets: a technique to analyze post-adhesion interaction between cancer cells and nets

Source: Kanamaru, R. et al. <a href="https://www.jove.com/video/58201" target="_blank">Neutrophil Extracellular Traps Generated by Low-density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment.</a> J. Vis. Exp. (2018)
In this video, we analyze the post-adhesion behavior of cancer cells trapped in neutrophil extracellular traps (NETs) by co-culturing low-density neutrophils and gastric cancer cells. The time-lapse video analysis helps monitor various cellular events and study the role of NETs in pathological conditions.

Time-lapse Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Analyze Post-adhesion Interaction between Cancer Cells and NETs

Source: Kanamaru, R. et al. Neutrophil Extracellular Traps Generated by Low-density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell ...

During pathological conditions like cancer, activated neutrophils release neutrophil extracellular traps or NETs - a network of extracellular DNA fibers associated with histones and proteolytic enzymes. These structures entrap cancer cells and play a key role in cancer metastasis.
To study the post-adhesion behavior of entrapped cancer cells in NETs in vitro, first, culture low-density neutrophils - a neutrophil subpopulation - in a polymer-coated culture dish that facilitates cell attachment. Incubate to activate the neutrophils to release mesh-like NETs.
Next, add a membrane-impermeable green fluorescent nuclear dye to stain the DNA fibers in NETs specifically. Remove the spent medium. Add a suspension of cancer cells to the neutrophil culture.
Incubate the culture for a short period to facilitate cancer cell-NET interaction. The DNA component of NETs functions as a chemotactic factor that stimulates the migration of cancer cells and aids in their adhesion to NETs.
Remove the spent medium. Gently rinse the culture with appropriate fresh media to remove any unbound cancer cells from the culture dish. Replenish with a fresh serum-containing media.
Image the culture for an extended duration using time-lapse video microscopy to analyze the interactive events between NETs and cancer cells. Over time, the NETs provide a suitable microenvironment facilitating the proliferation of cancer cells. Use suitable software to observe the proliferation of cancer cells on green fluorescent NETs.

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Research

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Assays and techniques

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

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

Time-lapse Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Analyze Post-adhesion Interaction between Cancer Cells and NETs

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