This study investigates the interaction between neutrophils and Staphylococcus aureus biofilms, focusing on reactive oxygen species (ROS) production. The methodology involves opsonization of biofilms and quantification of ROS using luminol in a multi-well plate setup.
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.
Add 100 microliters of 20% normal human serum diluted in HBSS, dropwise, to the washed biofilm, and incubate at 37 degrees Celsius for 30 minutes to opsonize the biofilm. Aspirate the serum solution, and wash the biofilms, dropwise, with 150 microliters of HBSS. Aspirate the HBSS, leaving behind wells with opsonized biofilms.
Add luminol to the neutrophils resuspended in HBSS to make up a final concentration of 5 micromolar luminol. This solution is ready to use for groups A and C. Add neutrophils mixed with luminol to the wells with opsonized biofilms.
For group D, prepare 50 micromolar luminol solution in HBSS in a separate tube without any neutrophils, and add it to the well containing the biofilm. Aliquot 350 microliters of neutrophils mixed with luminol, and add PMA at a final concentration of 500 nanograms per milliliter to the mixture.
For group B, add the neutrophils from this mixture into wells without biofilm. This serves as a positive control. Centrifuge the plate at 270 RCF for 30 seconds at 4 degrees Celsius. Ensure the plate reader is set to 37 degrees Celsius, the luminescence and kinetic read for 60 minutes with 3-minute intervals.
Place the plate in the plate reader to measure ROS production by neutrophils for 60 minutes.
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