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Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy
Published on: July 17, 2013
Studying interactions of Staphylococcus aureus with neutrophils by flow cytometry and time lapse microscopy
Bas G J Surewaard1, Jos A G van Strijp, Reindert Nijland
1Medical Microbiology, University Medical Center Utrecht, The Netherlands.
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 Ca(2+). 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.
Insights
We developed methods to study how Staphylococcus aureus toxins, specifically phenol soluble modulins (PSMs), activate neutrophils. This research quantifies PSM-induced neutrophil calcium signaling and expression, aiding in the development of targeted therapies.
Area of Science:
- Microbiology
- Immunology
- Toxicology
Background:
- Staphylococcus aureus produces phenol soluble modulins (PSMs) and other toxins.
- Neutrophils are key immune cells involved in combating bacterial infections.
- Understanding toxin-host interactions is crucial for developing effective treatments.
Purpose of the Study:
- To establish methods for studying the effects of PSMs on neutrophils.
- To quantify neutrophil activation by PSMs.
- To investigate the intracellular expression of PSMs during host-pathogen interactions.
Main Methods:
- Isolation of neutrophils via density gradient centrifugation.
- Measurement of intracellular calcium mobilization using fluorescent dyes and flow cytometry.
- Construction of reporter gene fusions (PSMα operon to GFP) for visualizing PSM expression in S. aureus.
Main Results:
- Developed a flow cytometry assay to measure PSM-induced neutrophil activation by monitoring calcium flux.
- Determined the concentration of PSMs required to activate neutrophils.
- Visualized intracellular PSM expression in S. aureus phagocytosed by neutrophils using fluorescence microscopy.
Conclusions:
- The presented methods allow for quantitative analysis of PSM-induced neutrophil responses.
- These techniques facilitate the study of PSM-neutrophil interactions and the evaluation of potential inhibitors.
- Reporter fusions provide insights into the regulation of PSM expression during infection.
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