eoe sub articles

EoE Sub Articles

1. NET-Cancer Cell Static Adhesion Assay
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	<li>Add 100 &#956;l of the previously obtained NET stock per well in a 96-well flat bottom plate and allow to coat wells O/N at 4 &#176;C in the dark.</li>
	<li>Between 12 and 20 hr later, verify the formation of a uniform monolayer of cell-free NETs at the bottom of the wells under the microscope (Figure 1). At this point, gently aspirate all non-adherent material out of the wells, making sure not to disrupt the NET monolayer at the botto...

in vitro adhesion assay to detect the adherence of cancer cells to neutrophil extracellular traps

Najmeh, S., et al. <a href="https://app.jove.com/v/52687">Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.</a> J. Vis. Exp. (2015)
In this video, we demonstrate an assay utilizing fluorescence microscopy to detect the adhesion of cancer cells to neutrophil extracellular traps (NETs). In the test wells, there is a notable increase in the number of fluorescently labeled cancer cells adhering to NETs, whereas wells treated with DNase exhibit reduced cancer cell adhesion to NETs.

<img alt="Figure 1" src="/files/ftp_upload/21804/21804_Figure1.jpg" /> 
Figure 1. NET Monolayer. Light microscopy images of the 96-well plate wells coated with NETs show (A) a uniform monolayer of NETs throughout the well as opposed to (B) inadequate coating of the well with an interrupted layer of NETs. Scale bars represent 40 &#956;m.
<img alt="Figure 2" src="/files/ftp_u...

In Vitro Adhesion Assay to Detect the Adherence of Cancer Cells to Neutrophil Extracellular Traps

1. NET-Cancer Cell Static Adhesion Assay

	Add 100 &#956;l of the previously obtained NET stock per well in a 96-well flat bottom plate and allow to coat ...

In the cancer microenvironment, activated neutrophils release neutrophil extracellular traps, or NETs, composed of extracellular DNA&#160; fibers, histones, and granular proteins, which play a crucial role in cancer progression.
To study cancer cell-NET adhesion in vitro, add NET suspension to multi-well plate wells. Incubate for NET monolayer formation.
Post-incubation, remove unadhered NETs.
Add a protein solution to block free binding sites in the wells.
Pipette fluorescently labeled cancer cells over NET monolayers.
Specific surface receptors on cancer cells interact with NETs, facilitating cell adhesion.
Post-incubation, discard media from the negative control well.
Add deoxyribonuclease to degrade NETs, detaching the cells.
Remove solutions from negative control and test wells. Use buffer to remove non-adherent cells.
Fix the adherent cancer cells to NETs with formaldehyde.
Using a fluorescence microscope, visualize the fluorescent cancer cells adhered to NETs.
Test wells show significant cancer cell adhesion to NETs, while deoxyribonuclease-treated NETs exhibit reduced levels of cancer cell adhesion.