Determination of ROS Production by Neutrophils upon Interaction with Opsonized Biofilm

Published: September 29, 2023

Abstract

Source: Rana, P. S. J. B., et al. Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms. J. Vis. Exp. (2022)

In this video, we demonstrate an in vitro chemiluminescence assay to detect the generation of reactive oxygen species by neutrophils upon exposure to an opsonized bacterial biofilm.

Protocol

1. Measurement of ROS produced by neutrophils 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. 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 recommen…

Divulgations

The authors have nothing to disclose.

Materials

50 mL conical centrifuge tubes Thermo Scientific 339652
Alcohol swab BD Used for blood draw
Cell counter Bal Supply 202C
Clear bottom 96-well flat bottom polystyrene plates Costar 3370
Dulbecco's phosphate-buffered saline (DPBS) 1x Gibco 14190-144
Hanks' balanced salt solution (HBSS) 1x Corning cellgro 21-022-CV Without calcium, magnesium, and phenol red
Hemocytometer Bright Line
Luminol Sigma A8511-5G
Minimal essential media (MEM) Alpha 1x Gibco 41061-029
Normal human serum Complement Technology NHS
Petri Dish (100 x 15 mm) VWR 25384-342
Phorbol 12-myristate 13-acetate
Poly-L-lysine solution Sigma P4707-50ML
SoftMax Pro Software Molecular Devices Microplate reader software used for data acquisition
SpectraMax i3x Molecular Devices Microplate reader

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Determination of ROS Production by Neutrophils upon Interaction with Opsonized Biofilm. J. Vis. Exp. (Pending Publication), e21642, doi: (2023).

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