Determination of ROS Production by Neutrophils upon Interaction with Opsonized Biofilm

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.