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Flow cytometry distinction between adherent and phagocytized yeast particles
J Giaimis1, Y Lombard, P Poindron
1Département d'Immunologie, Immunopharmacologie et Pathologie, Université Louis Pasteur de Strasbourg, France.
Cytometry
|October 1, 1994
Summary
This study introduces a novel flow cytometry method to differentiate between yeast on macrophage surfaces and those inside cells. This technique accurately quanties phagocytosis by distinguishing extracellular from intracellular yeast.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Distinguishing between surface-bound and phagocytized yeast is crucial for understanding host-pathogen interactions.
- Traditional methods can be time-consuming and lack high-throughput capabilities.
Purpose of the Study:
- To adapt a light microscopy staining technique for flow cytometry to quantify yeast phagocytosis by macrophages.
- To develop a rapid and unambiguous method for distinguishing extracellular from intracellular yeast.
Main Methods:
- Utilized fluorescent probes and a novel staining technique adapted for flow cytometry.
- Employed tannic acid solution to quench fluorescence of extracellular yeast.
- Used FITC-labeled, heat-killed yeast cells and macrophages.
Main Results:
- Successfully differentiated extracellular from intracellular yeast with high throughput (approx. 200 cells/s).
- Demonstrated that tannic acid effectively quenches fluorescence of external yeast while internal yeast retain fluorescence.
- Validated the technique by showing inhibition of endocytosis by cytochalasin-B.
Conclusions:
- The developed flow cytometry technique provides a robust and efficient method for assessing phagocytic activity.
- This approach offers a significant advancement for studying host-pathogen interactions and immune responses.