Determination of ROS Production by Neutrophils upon Interaction with Opsonized Biofilm

1. Measurement of ROS produced by neutrophils

1. Add 100 µL of 20% normal human serum (diluted in HBSS) dropwise to the washed biofilm and incubate at 37 °C under static conditions for 30 min to opsonize the biofilm.
2. Aspirate the 20% serum solution and wash the biofilms dropwise with 150 µL of HBSS once. Aspirate the HBSS, leaving behind wells with opsonized biofilms.
   NOTE: For interpretation of the experiment, a minimum of four groups is recommended: (A) Neutrophils + Biofilm, (B) Neutrophils + PMA (positive control, see Table of Materials), (C) Neutrophils only, and (D) Biofilm only.
3. Add luminol (see Table of Materials) to the neutrophils resuspended in HBSS at a concentration of 4 x 10⁶ cells/mL such that the final luminol concentration is 50 µM. This solution is ready to use for groups (A) and (C). Add 4 x 10⁵ neutrophils mixed with luminol to the wells with opsonized biofilms.
4. In a separate tube, prepare 50 µM luminol solution in HBSS without any neutrophils and add it to the well containing biofilm (group D).
5. Aliquot 350 µL of neutrophils mixed with luminol and add phorbol 12-myristate 13-acetate (PMA) at a final concentration of 500 ng/mL to the mixture. For group (B), add 4 x 10⁵ neutrophils from this mixture into wells without biofilm. This serves as a positive control.
   NOTE: The concentration of PMA indicated in this step is relatively high to ensure a robust burst response as PMA-stimulated neutrophils are a positive control. PMA can be used at a lower concentration to activate neutrophils, depending on the experiment.
6. Centrifuge the plate at 270 x g for 30 s at 4 °C.
7. Ensure the plate reader is set to 37 °C along with the setting for luminescence and kinetic read for 60 min with 3 min intervals. Place the plate in the plate reader to measure ROS production by neutrophils for 60 min.
   NOTE: For this assay, biofilms were grown in white plates used for luminescence assays. PMA is a known agonist for the oxidative burst response. When performing studies involving PMA, ensure that PMA is added at the final step while the solution containing neutrophils is cold since PMA immediately initiates the burst response.