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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.
Abstract:
Our laboratory recently developed a light microscopy staining technique that provides a mean to distinguish between yeast that are simply bound to the surface of macrophages and yeast that have actually been phagocytized by macrophages (7). We adapted this technique by using fluorescent probes in order to test phagocytic activity by flow cytometry. Thus we are able to distinguish unambiguously extracellular from intracellular yeast during phagocytosis with the fast rate of flow cytometry (approximately 200 cells/s). The fluorescence quenching induced by a 1% tannic acid solution (w/v) can be applied to any FITC-labeled, heat-killed yeast cell or bacteria. The yeast cells already engulfed in the macrophage remain with their native fluorescence (internal and external pH equilibrated by 50 microM monensin 30 min/4 degrees C) protected from the action of tannic acid, a nonmembrane permeable molecule. The results presented here validate this new technique. An application is presented showing the inhibition of endocytosis by cytochalasin-B.
Insights
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.