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https://www.arca.fiocruz.br/handle/icict/66325
PROTOCOL FOR SEPARATION OF FUNGAL EXTRACELLULAR VESICLES USING ULTRACENTRIFUGATION FROM SOLID MEDIUM CULTURES
Author
Affilliation
Institut Pasteur. Université Paris Cité. Unité Biologie des ARN des Pathogénes Fongiques. Département de Mycologie. Paris, France.
Institut Pasteur. Université Paris Cité. Unité Biologie des ARN des Pathogénes Fongiques. Département de Mycologie. Paris, France.
Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.
Institut Pasteur. Université Paris Cité. Unité Biologie des ARN des Pathogénes Fongiques. Département de Mycologie. Paris, France.
Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.
Institut Pasteur. Université Paris Cité. Unité Biologie des ARN des Pathogénes Fongiques. Département de Mycologie. Paris, France.
Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.
Institut Pasteur. Université Paris Cité. Unité Biologie des ARN des Pathogénes Fongiques. Département de Mycologie. Paris, France.
Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.
Abstract
Extracellular vesicles (EVs) have been identified in diverse fungi, including human pathogens. In this protocol, we present two techniques for isolating and analyzing fungal EVs. The first is for high-throughput screening, and the second is for yielding concentrated samples suitable for centrifugation-based density gradients. We describe steps for analytical assays such as nano-flow cytometry and nanoparticle tracking analysis to measure EV dimensions and concentration. EV suspensions can serve diverse assays, including electron microscopy, compo sitional determination, and cell-to-cell communication assays. For complete details on the use and execution of this protocol, please refer to Rizzo et al.,1 Rizzo et al.,2 Reis et al.,3 and Reis et al.4.
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