eoe sub articles

EoE Sub Articles

1.&#160;S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density at 600 nm (OD600) and concentration of bacteria must be determined empirically for the S. aureus strains to be tested before beginning this assay. The success of these experiments requires the consistent harvest of equal concentrations of each S. aureus</e...

cytotoxicity assay of staphylococcus aureus strains on neutrophils following phagocytosis

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)
In this video, we demonstrate an assay to detect the cytotoxicity of virulent and less virulent Staphylococcus aureus strains against human neutrophils following phagocytosis using flow cytometry.

<img alt="Figure 1" src="/files/ftp_upload/21790/21790_Figure1.jpg" /> 
Figure 1: Flow cytometry analysis of PMNs following phagocytosis of S. aureus. (A) The proportion of propidium iodide positive PMNs 90 min after the phagocytosis of different concentrations of USA300 or USA300&#8710;saeR/S. (B) The concentration of opsonized S. aureus strains used for the experiments is shown in panel A. Data are presented as mean &#177;...

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

1.&#160;S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density ...

Take virulent and less virulent Staphylococcus aureus strains.
Add serum to opsonize the bacteria.
Transfer increasing bacterial concentrations to chilled multi-well plate wells containing neutrophils. Low temperatures prevent neutrophil activation.
Centrifuge to facilitate synchronized phagocytosis.
Neutrophil receptors bind the opsonins, triggering bacterial internalization into phagosomes.
In virulent bacteria, the phagosomal environment triggers the expression of cytolytic toxins.
These toxins form pores, disrupting the phagosomes and compromising the neutrophil membrane integrity.
However, the less virulent bacteria exhibit reduced cytolytic toxin expression.
Post-incubation, detach the neutrophils and transfer them to flow cytometer tubes containing fluorescent propidium iodide (PI) dye.
The dye enters the neutrophils with compromised membranes and stains the DNA.
Using a flow cytometer, analyze the neutrophils for PI staining.
A concentration-dependent increase in the proportion of PI-positive neutrophils following phagocytosis of the virulent strain, as compared to the less virulent strain, indicates the cytotoxicity of the virulent strain against neutrophils.