eoe sub articles

EoE Sub Articles

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. Isolation of LDN from Abdominal Cavity Lavages 
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	<li>Sample acquisition
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		<li>Infuse 1,000 mL of normal sterile saline directly into the abdominal cavity before wound closure in patients who have undergone abdominal surgery due to gast...

peritoneal low-density neutrophil isolation: a technique to obtain low-density neutrophils from peritoneal lavage fluid using magnetic activated cell sorting

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 describe the procedure for isolating low-density neutrophils, or LDNs, from the peritoneal lavage fluid of a postoperative human patient. The LDNs thus obtained can be used in further downstream experiments to understand their role in tumor growth and progression.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20414/20414_Figure1.jpg" /> 
Figure 1: NET formation after the culture of peritoneal LDN. (A)&#160;After density gradient centrifugation of postoperative lavage fluid, cells recovered from the intermediate layer are shown.&#160;(B)&#160;Then, CD66b(+) LDN were separated from CD66b(-) mononuclear cells and cultured on poly-L-lysine coated 6-well plates, followed by nuclear staining dye additio...

Peritoneal Low-density Neutrophil Isolation: A Technique to Obtain Low-density Neutrophils from Peritoneal Lavage Fluid Using Magnetic Activated Cell Sorting

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

Low-density neutrophils, or LDNs, are a distinct neutrophil subpopulation found in elevated numbers during pathological conditions like gastrointestinal cancer.&#160; LDNs can be isolated from the peritoneal cavity washes or lavage fluid of patients.
First, filter the lavage fluid to remove tissue debris. Centrifuge the filtrate to obtain a pellet containing blood cells, including LDNs. Resuspend it in a suitable buffer. Layer this suspension over a suitable density gradient medium optimal for LDN isolation.
Centrifuge to separate the different blood components based on their relative densities. The denser RBCs form the bottom layer, while the least dense plasma fraction forms the topmost layer. The less-dense LDNs occupy the intermediate layer along with other mononuclear cells.
Transfer the intermediate layer into a fresh tube containing a blocking reagent. The reagent components block non-specific binding sites on all non-target cells&#39; surfaces and increase the specificity for LDN during subsequent labeling.
Add antibody-conjugated microbeads targeting a specific cell surface molecule overexpressed on LDNs. Incubate for the antibody-bead complexes to bind to their target molecules on LDNs.
Pass the incubated suspension through a column placed in a magnetic field. Under the magnetic influence, all LDN-microbead complexes adhere to the column wall while unlabeled cells pass through.
Remove the column. Flush the contents using a suitable buffer to elute and collect the LDN-containing fraction.

Peritoneal Low-density Neutrophil Isolation: A Technique to Obtain Low-density Neutrophils from Peritoneal Lavage Fluid Using Magnetic Activated Cell Sorting

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