This study investigates the cytotoxic effects of virulent and less virulent strains of Staphylococcus aureus on neutrophils. The research focuses on how these strains interact with neutrophils during phagocytosis and the subsequent effects on neutrophil membrane integrity.
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.
When subcultured Staph aureus has reached mid-exponential growth, transfer 1 milliliter of cultured bacteria to 1.5-milliliter microcentrifuge tube, and centrifuge at 5000 g for 5 minutes at room temperature. Wash S. aureus by aspirating supernatant without disturbing the pellet, and resuspend the pelleted bacteria in 1 milliliter DPBS. Vortex the samples for 30 seconds. Centrifuge the samples at 5000 g for 5 minutes at room temperature.
To opsonize Staph aureus, first, resuspend the bacterial pellet in 1 milliliter of 20% human serum. Then, incubate samples at 37 degrees Celsius with agitation for 15 minutes. Wash opsonized Staph aureus following centrifugation, as shown previously. Dilute opsonized Staph aureus strains to 1 x 108 colony-forming units, CFUs per milliliter, with ice-cold RPMI. Vortex the sample for 30 seconds, and place on ice.
Confirm the concentrations of Staph aureus used for these assays by plating 1 to 10 serial dilutions of bacteria on agar. Gently, add 100 microliters per well of each Staph aureus strain, or RPMI, for positive and negative controls to PMNs in a 96-well plate on ice from step 1.14. Gently rock plate to distribute Staph aureus in wells.
Synchronize phagocytosis by centrifuging plate at 500 g for eight minutes at 4 degrees Celsius, and incubate plate at 37 degrees Celsius, immediately following centrifugation as time 0. At the desired time points, put the plate on ice, and add 200 microliters of ice-cold DPBS containing 1 microliter of propidium iodide to each flow cytometry tube, then, transfer samples to their corresponding tube.
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