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Isolation and Characterization of Cyanobacterial Extracellular Vesicles
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Isolation and Characterization of Cyanobacterial Extracellular Vesicles

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Protocol for separation of fungal extracellular vesicles using ultracentrifugation from solid medium cultures

Alicia C Piffer1, Flavia C G Reis2, Frédérique Moyrand1

  • 1Institut Pasteur, Université Paris Cité, Unité Biologie des ARN des Pathogènes Fongiques, Département de Mycologie, 75015 Paris, France.

STAR Protocols
|May 21, 2024
PubMed

Insights

This protocol details methods for isolating and analyzing fungal extracellular vesicles (EVs), crucial for understanding fungal pathogens. These techniques enable high-throughput screening and concentrated sample preparation for various downstream applications.

Area of Science:

  • Mycology
  • Cell Biology
  • Biotechnology

Background:

  • Extracellular vesicles (EVs) are increasingly recognized in fungal biology, including in human pathogenic species.
  • Understanding fungal EVs is vital for developing novel diagnostic and therapeutic strategies.

Purpose of the Study:

  • To present two distinct, optimized protocols for the isolation and analysis of fungal extracellular vesicles (EVs).
  • To provide a comprehensive guide for researchers working with fungal EVs, facilitating further investigation into their roles.

Main Methods:

  • Description of a high-throughput screening method for rapid fungal EV isolation.
  • Detailed protocol for obtaining concentrated fungal EV samples suitable for density gradient ultracentrifugation.
  • Guidance on analytical assays including nano-flow cytometry and nanoparticle tracking analysis for EV characterization.

Main Results:

  • The presented methods allow for efficient isolation of fungal EVs from diverse fungal species.
  • Analytical techniques enable accurate measurement of EV size distribution and concentration.
  • EV suspensions are suitable for a wide range of downstream applications, including microscopy and compositional analysis.

Conclusions:

  • The developed protocols offer robust and versatile approaches for fungal EV research.
  • These methods will aid in advancing the study of fungal EVs, their biogenesis, and their functional roles in health and disease.
  • Standardized isolation and analysis techniques are essential for comparative studies across different fungal pathogens.
Keywords:
Cell BiologyCell isolationCell-based AssaysFlow CytometryMicrobiologyModel Organisms

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