Use a fluorescent strain of S. aureus, such as USA300 expressing GFP, to ease microscopy imaging. Incubate neutrophils with 100 micromolar BCD for 30 minutes in a rocker at 37 degrees Celsius with 5% atmospheric carbon dioxide. Ensure the samples are incubated in the dark and limit light exposure.
To wash excess BCD, centrifuge neutrophils at 270 RCF for five minutes, and aspirate the supernatant. Resuspend the neutrophils in fresh HBSS. Then, add ethidium homodimer-1 to the BCD-stained neutrophils at a final concentration of 4 micromolar to monitor neutrophil and bacterial death.
Wash the biofilm with HBSS, and add 150 microliters of neutrophils to the S. aureus biofilm that has been grown in microslides. Incubate the microslides in a humidified chamber for 30 minutes. The number of bacterial cells will be based on the cell counts obtained from the 18-hour biofilm plating.
Image the neutrophil biofilm interaction using fluorescent channels corresponding to fluorescent dyes or proteins' excitation and emission wavelengths.
