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. Purification of human polymorphonuclear leukocytes and isolation of human serum
NOTE: All reagents should be routinely checked for the presence of endotoxin using a commercially available endotoxin detection kit ...

an assay for bacterial extracellular protein-mediated cytotoxicity on pmns

Source: Dankoff, J. G., et al. <a href="https://app.jove.com/v/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.

<img alt="Figure 1" src="/files/ftp_upload/21789/21789_Figure1.jpg" /> 
Figure 1: Flow cytometry analysis of purified PMNs. Representative flow cytometry dot plots of (A) purified human PMNs and (B) PMNs that have been purposely contaminated with peripheral blood mononuclear cells. (C) Representative flow cytometry histogram demonstrating minimal propidium iodide staining (&#60;1%) of purified PMNs (shaded grey) as compared to PMNs treated with 0.05% Triton X-100 (sh...

An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Take a culture of Staphylococcus aureus, a pathogenic bacteria that secretes leukocidin&#160; &#8212; a pore-forming toxin &#8212; comprising monomeric&#160; S and F components.
Pellet the cells and serially dilute the supernatant. Transfer the leukocidin-containing supernatant into a microplate having polymorphonuclear leukocytes, or PMNs, and incubate.
The S component binds to specific transmembrane receptors on PMNs, followed by hetero-oligomerization with the F component to insert into the cell membrane, resulting in pore formation.
The pore causes a decrease in intracellular potassium ions, activating NLR family pyrin domain-containing 3 or NLRP3 &#160;protein.
NLRP3 binds to apoptosis-associated speck-like protein containing a caspase recruitment domain, or ASC, which recruits pro-caspase-1, forming an inflammasome.
Auto-proteolysis converts pro-caspase-1 to active caspase-1, triggering cell death.
Harvest the cells at defined intervals. Add propidium iodide or PI, which enters dead cells through damaged membranes to bind DNA, emitting fluorescence.
Using flow cytometry, quantify PI-stained cells to assess the cytotoxic effect of both leukocidin concentration and incubation time.