An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

Culture S. aureus overnight in appropriate media, using a shaking incubator set at 37 degrees Celsius the day prior to the experiment. Subculture S. aureus by performing a 1 to 100 dilution of overnight bacteria cultures with fresh media. Incubate at 37 degrees Celsius with shaking until bacteria reach early stationary growth phase. After five hours of growth, transfer 1 milliliter of subculture S. aureus into a 1.5-milliliter microcentrifuge tube, and centrifuge at 5000 g for 5 minutes at room temperature.

Following centrifugation, aspirate supernatants. Pass aspirated supernatants through a 0.22-micron syringe filter into a new 1.5-milliliter microcentrifuge tube, and place on ice. Perform serial dilutions of supernatant with ice-cold media used to culture Staph aureus. Gently add supernatant samples or media alone for negative and positive controls to individual wells of 96-well plate containing PMNs and ice as described earlier.

Gently rock plate to distribute supernatants and wells, and incubate at 37 degrees Celsius. At desired times, remove plate from incubator, and place on ice. Add 300 microliters of ice-cold DPBS containing 1 microliter of Propidium iodide to each flow cytometry tube. Transfer the samples to flow cytometry tubes on ice. For the positive control well, add 20 microliters of 10% Triton X to the sample prior to transferring to a flow cytometry tube. Analyze propidium iodide staining of intoxicated PMNs using flow cytometry.