Determination of ROS Production by Neutrophils upon Interaction with Opsonized Biofilm

Bacteria tagged with opsonins are recognized by specific neutrophil receptors, triggering phagocytosis with reactive oxygen species, or ROS, release, and bacterial degradation.

To quantify ROS production by neutrophils in vitro, take a multi-well plate. The test well contains a Staphylococcus aureus biofilm comprising extracellular matrix film-embedded bacteria. The control well is without the biofilm.

Incubate the biofilm with human serum, allowing serum opsonins to opsonize the biofilm.

Aspirate the serum. Wash with buffer, removing unbound opsonins. Add neutrophils and luminol — a chemiluminescent compound to the wells. Centrifuge, bringing the neutrophils near the biofilm.

Using a plate reader, measure the luminescence over time.

In the biofilm-containing well, neutrophils bind to the biofilm's opsonins, causing neutrophil activation and bacterial internalization into phagosomes. Specific signaling pathways get activated, causing NADPH oxidase assembly on phagosomes. 

NADPH oxidase catalyzes electron transfer from NADPH to molecular oxygen, generating ROS that degrade bacteria. Further, NADPH oxidase on the cell membrane releases ROS extracellularly.

In response, biofilm bacteria produce enzymes that neutralize ROS and release leukocidins that form pores in neutrophil membranes and cause lysis, disrupting ROS production. Luminol reacts with ROS, causing its excitation, and emits luminescence upon relaxation.

The test well exhibits increased ROS levels compared to the control, indicating neutrophil-opsonized biofilm interactions.