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 ±…
Divulgations
The authors have nothing to disclose.
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