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