Source: Dankoff, J. G. et al., Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes. J. Vis. Exp. (2020)
In this video, we demonstrate an assay to detect the cytotoxicity of virulent and less virulent Staphylococcus aureus strains against human neutrophils following phagocytosis using flow cytometry.
1. S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density at 600 nm (OD600) and concentration of bacteria must be determined empirically for the S. aureus strains to be tested before beginning this assay. The success of these experiments requires the consistent harvest of equal concentrations of each S. aureus strain tested at the mid-exponential growth phase using the OD600 of sub-cultured bacteria.
Figure 1: Flow cytometry analysis of PMNs following phagocytosis of S. aureus. (A) The proportion of propidium iodide positive PMNs 90 min after the phagocytosis of different concentrations of USA300 or USA300∆saeR/S. (B) The concentration of opsonized S. aureus strains used for the experiments is shown in panel A. Data are presented as mean ± SEM of 4 separate experiments with * p ≤ 0.01 as determined by the two-tailed t-test.
The authors have nothing to disclose.
1.5 mL micro-centrifuge tubes with Snap Caps | VWR | 89000-044 | Used in washing cells |
12x75mm Culture tubes | VWR | 60818-430 | Used as flow cytometry tubes |
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 |
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 |
Triton X-100 | Sigma-Aldrich | X100 | Membrane integrity positive control |