Determination of ROS Production by Neutrophils upon Interaction with Opsonized Biofilm

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

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) Biofi....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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