Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

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.