Protocol for cross-platform characterization of human and murine extracellular vesicles and particles

Linda Bojmar; Han Sang Kim; Kei Sugiura; Søren Heissel; Serena Lucotti; Michele Cioffi; Kofi Ennu Johnson; Leona Cohen-Gould; Haiying Zhang; Henrik Molina; Irina R Matei; David Lyden; Ayuko Hoshino

doi:10.1016/j.xpro.2023.102754

Protocol for cross-platform characterization of human and murine extracellular vesicles and particles

STAR Protoc. 2024 Mar 15;5(1):102754. doi: 10.1016/j.xpro.2023.102754. Epub 2023 Dec 13.

Authors

Linda Bojmar¹, Han Sang Kim², Kei Sugiura³, Søren Heissel⁴, Serena Lucotti⁵, Michele Cioffi⁵, Kofi Ennu Johnson⁶, Leona Cohen-Gould⁷, Haiying Zhang⁵, Henrik Molina⁴, Irina R Matei⁸, David Lyden⁹, Ayuko Hoshino¹⁰

Affiliations

¹ Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.
² Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Graduate School of Medical Science, Brain Korea 21 FOUR Project, Yonsei University College of Medicine, Seoul, Korea.
³ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan; School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
⁴ Proteomics Resource Center, The Rockefeller University, New York, NY, USA.
⁵ Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.
⁶ Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Tri-Institutional PhD Program in Computational Biology and Medicine, New York, NY, USA.
⁷ Microscopy & Image Analysis, Core Facilities, Weill Cornell Medicine, New York, NY, USA.
⁸ Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. Electronic address: irm2224@med.cornell.edu.
⁹ Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. Electronic address: dcl2001@med.cornell.edu.
¹⁰ Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan; School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan. Electronic address: aynhoshino@g.ecc.u-tokyo.ac.jp.

Abstract

Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross-validation across different platforms. For complete details on the use and execution of this protocol, please refer to Hoshino et al.¹.

Keywords: Cell Biology; Cell Membrane; Cell isolation; Mass Spectrometry; Model Organisms; Molecular Biology; Protein Biochemistry; Proteomics.

MeSH terms

Animals
Blotting, Western
Computational Biology
Extracellular Vesicles*
Humans
Mass Spectrometry
Mice
Proteomics*

Abstract

MeSH terms

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