An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs
Published: November 30, 2023
Abstract
Source: Dankoff, J. G., et al. Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes. J. Vis. Exp. (2020).
This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Purification of human polymorphonuclear leukocytes and isolation of human serum NOTE: All reagents should be routinely checked for the presence of endotoxin using a commercially available endotoxin detection kit …
Representative Results
Figure 1: Flow cytometry analysis of purified PMNs. Representative flow cytometry dot plots of (A) purified human PMNs and (B) PMNs that have been purposely contaminated with peripheral blood mononuclear cells. (C) Representative flow cytometry histogram demonstrating minimal propidium iodide staining (<1%) of purified PMNs (shaded grey) as compared to PMNs treated with 0.05% Triton X-100 (sh…
Divulgations
The authors have nothing to disclose.
Materials
| 0.9% Sodium Chloride Injection, USP, 500 mL VIAFLEX Plastic Container | Baxter | 2B1323Q | PMN purification |
| 1.5 mL micro-centrifuge tubes with Snap Caps | VWR | 89000-044 | Used in washing cells |
| 1.8% Sodium Chloride Solution | Sigma-Aldrich | S5150 | PMN purification |
| 12x75mm Culture tubes | VWR | 60818-430 | Used as flow cytometry tubes |
| 20% (w/w) Dextran | Sigma-Aldrich | D8802 | PMN purification |
| 3125 Hand Tally Counter | Traceable Products | 3125CC | For counting cells |
| 50 mL conical centrifuge tubes | VWR | 89039-656 | For dispensing media |
| Bacto Tryptic Soy Broth, Soybean-Casein Digest Medium | FischerScientific | 211823 | For growing cell cultures |
| BD Disposable Syringes with Luer-Lok Tips, 3 mL | FischerScientific | BD 309657 | For filtering supernatants |
| Bright-Line Hemocytometer | Sigma-Aldrich | Z359629 | Cell counting apparatus |
| DPBS, 1x (Dulbecco's Phosphate Buffered Saline) with calcium and magnesium | Corning | 21-030-CV | Used in washing cells |
| Ficoll-Paque PLUS | GE Healthcare | 17-1440-02 | PMN purification |
| Fisherbrand Sterile Polystyrene Disposable Serological Pipets | FischerScientific | 13-678-11E | For aspirating liquid |
| Greiner CELLSTAR 96 well plates | Millipore Sigma | M0687 | Plate for holding experimental samples |
| OMICRON Syringe Filters | Omicron Scientific | SFPV13R | For filtering supernatants |
| Propidium iodide | ThermoFisher Scientific | P3566 | Membrane impermeable DNA stain |
| PYREX Brand 4980 Erlenmeyer Flasks | Cole-Parmer | EW-34503-24 | For growing cell cultures |
| RPMI 1640, 1X without L-glutamine, phenol red | Corning | 17-105-CV | Used in resuspending cells |
| Sterile Water for Irrigation, USP | Baxter | 2F7113 | PMN purification |
| The Pipette Pump | Bel-Art Products | F37898 | For aspirating liquid |
| Triton X-100 | Sigma-Aldrich | X100 | Membrane integrity positive control |
Citer Cet Article
An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs. J. Vis. Exp. (Pending Publication), e21789, doi: (2023).