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Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy |
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| Authors: | Bas G.J. Surewaard Jos A.G. van Strijp Reindert Nijland |
| Affiliation: | 1.Medical Microbiology, University Medical Center Utrecht |
| Abstract: | We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy. |
| Keywords: | Infection Issue 77 Immunology Cellular Biology Infectious Diseases Microbiology Genetics Medicine Biomedical Engineering Bioengineering Neutrophils Staphylococcus aureus Bacterial Toxins Microscopy Fluorescence Time-Lapse Imaging Phagocytosis phenol soluble modulins PSMs Polymorphonuclear Neutrophils PMNs intracellular expression time-lapse microscopy flow cytometry cell isolation cell culture |