Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

Abstract

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.

Protocol

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</e…

Representative Results

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 ±…

Materials

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