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Purification of High Yield Extracellular Vesicle Preparations Away from Virus
Published on: September 12, 2019
Isolation and Purification of Extracellular Vesicles from Aspergillus fumigatus
Lucas F B Nogueira1, Fausto Almeida2
1Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
Abstract:
Extracellular vesicles (EVs) are increasingly recognized as key players in fungal biology, mediating intercellular communication, modulating virulence, and shaping host-pathogen interactions. Despite their growing importance, the lack of standardized protocols for EV isolation in fungi has hindered reproducibility and the accurate interpretation of functional data. This chapter presents a comprehensive, optimized protocol for the isolation and characterization of EVs produced by Aspergillus fumigatus under both solid and liquid culture conditions. Culture parameters, including medium composition and growth format, are critically evaluated for their impact on EV yield, molecular composition, and biological significance. The protocol encompasses detailed procedures for fungal cultivation, EV isolation via differential ultracentrifugation, and multiparametric characterization through nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and molecular marker profiling, all aligned with MISEV2023 guidelines. Designed to support both exploratory and high-resolution investigations, this standardized approach robustly analyzes EV-associated biomarkers. The isolated EVs are suitable for a range of downstream applications, including studies of immune modulation, molecular composition, fungal pathogenesis, and intercellular signaling dynamics.
Insights
This study provides a standardized protocol for isolating fungal extracellular vesicles (EVs) from Aspergillus fumigatus. This method enhances reproducibility for studying fungal pathogenesis and intercellular communication.
Area of Science:
- Mycology
- Cell Biology
- Biochemistry
Background:
- Extracellular vesicles (EVs) are crucial for fungal intercellular communication, virulence, and host-pathogen interactions.
- Standardized protocols for fungal EV isolation are lacking, hindering research reproducibility and data interpretation.
Purpose of the Study:
- To present a comprehensive, optimized protocol for isolating and characterizing EVs from Aspergillus fumigatus.
- To evaluate culture parameters influencing EV yield, composition, and biological significance.
- To establish a standardized approach for robust EV biomarker analysis.
Main Methods:
- Detailed procedures for fungal cultivation (solid and liquid media) and EV isolation using differential ultracentrifugation.
- Multiparametric characterization via Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and molecular marker profiling.
- Adherence to Minimum Information for Studies of Extracellular Vesicles 2023 (MISEV2023) guidelines.
Main Results:
- An optimized protocol for Aspergillus fumigatus EV isolation and characterization is presented.
- Culture parameters critically impact EV yield, molecular composition, and biological relevance.
- The protocol enables robust analysis of EV-associated biomarkers.
Conclusions:
- This standardized protocol facilitates reproducible isolation and characterization of fungal EVs.
- The characterized EVs are suitable for diverse downstream applications, including immune modulation and pathogenesis studies.
- The approach supports both exploratory and high-resolution investigations into fungal biology.
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