Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

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.