An In Vitro Assay to Study the Interaction between Neutrophils and Biofilm

A biofilm is a community of surface-adhered bacteria embedded inside a matrix of extracellular polymeric substances, EPS, of microbial origin.

To study immune cell-biofilm interactions in vitro, take a slide with a channel containing a biofilm of Staphylococcus aureus — an opportunistic pathogen. The engineered bacteria express a fluorescent protein for microscopic detection. The channel connects to reservoirs filled with media to supply nutrients to the bacteria.

Add a suspension of neutrophils labeled with a fluorescent cell-tracking dye. The medium also contains an ethidium homodimer, which stains the dead cells' DNA. Incubate under physiological conditions.

The pattern recognition receptors on neutrophils bind to the pathogen-associated molecular patterns on the bacteria. The binding induces neutrophils to phagocytose the bacteria and produce extracellular reactive oxygen species or ROS, killing the bacteria.

To evade the immune response, bacteria release detoxifying enzymes that diminish the ROS and leukocidin toxin that causes cell death. Monomeric leukocidin binds to specific receptors on the neutrophils and undergoes multimerization to form a pore, disrupting membrane integrity and killing the cells.

The ethidium homodimer enters through the damaged cell membrane, staining the dead cells.

Under a wide-field fluorescence microscope, a subset of bacterial cells and neutrophils appear dead, indicating neutrophil-biofilm interaction.

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.