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Published on: June 30, 2023
Quantitation of microsporidia in cultured cells by flow cytometry
Caspar Franzen1, Andreas Müller, Pia Hartmann
1Department of Internal Medicine I, University of Regensburg, Regensburg, Germany. Caspar.Franzen@Klinik.Uni-Regensburg.de
Background:
Microsporidia are obligate intracellular protozoan parasites that emerged as major opportunistic pathogens in humans since the onset of the AIDS pandemic. In the present study, we investigated whether FCM is a useful method for the quantitation of intracellular microsporidian spores in cultured cells.
Methods:
Microsporidia (Encephalitozoon cuniculi) were grown in cell cultures and various cell-lines were coincubated with microsporidian spores at different multiplicities of infection, as well as for different periods of time. After permeabilization of the cells, intracellular spores were stained with a polyclonal anti-E. cuniculi serum and a FITC-labeled secondary antibody. Stained cells were analyzed on a flow cytometer and results were compared with those of fluorescence microscopy.
Results:
Noninfected cells showed a lower fluorescence, while the relative fluorescence observed for infected cells was significantly higher. The cell population with the more intense fluorescence, representing cells with internalized microsporidian spores, increased with the multiplicity of infection as well as over time. Results of FCM and fluorescence microscopy were in excellent agreement for all experiments.
Conclusions:
We have developed a flow cytometric assay to detect and quantify cells with intracellular microsporidian spores. This method is easy to use, highly reproducible, and should be useful for future research.
Insights
Flow cytometry (FCM) effectively quantifies intracellular microsporidian spores in cultured cells. This reproducible assay aids in studying microsporidian infections, particularly Encephalitozoon cuniculi.
Area of Science:
- Parasitology
- Cell Biology
- Immunology
Background:
- Microsporidia are opportunistic pathogens, especially in immunocompromised individuals.
- The AIDS pandemic increased the clinical significance of microsporidian infections.
- Accurate quantification of intracellular microsporidia is crucial for research.
Purpose of the Study:
- To evaluate flow cytometry (FCM) for quantifying intracellular microsporidian spores.
- To establish a reliable method for detecting microsporidian presence in host cells.
Main Methods:
- Encephalitozoon cuniculi spores were used to infect various cell lines.
- Intracellular spores were stained using immunofluorescence with anti-E. cuniculi antibodies and FITC.
- Flow cytometry and fluorescence microscopy were employed for analysis and comparison.
Main Results:
- Infected cells exhibited significantly higher fluorescence compared to uninfected cells.
- Increased fluorescence correlated with higher multiplicities of infection and longer incubation times.
- Flow cytometry results showed excellent agreement with fluorescence microscopy.
Conclusions:
- A flow cytometric assay was successfully developed for detecting and quantifying intracellular microsporidian spores.
- The developed FCM method is user-friendly, highly reproducible, and valuable for future research.
- This assay provides a robust tool for studying microsporidian pathogenesis and host-cell interactions.
