A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture

STAR Protoc. 2023 Dec 15;4(4):102562. doi: 10.1016/j.xpro.2023.102562. Epub 2023 Sep 9.

Abstract

Traditionally, midbody remnants (MBRs) are isolated from cell culture medium using ultracentrifugation, which is expensive and time consuming. Here, we present a protocol for isolating MBRs or large extracellular vesicles (EVs) from mammalian cell culture using either 1.5% polyethylene glycol 6000 (PEG6000) or PEG5000-coated gold nanoparticles. We describe steps for growing cells, collecting media, and precipitating MBRs and EVs from cell culture medium. We then detail characterization of MBRs through immunofluorescent antibody staining and immunofluorescent imaging.

Keywords: Cancer; Cell Biology; Cell Culture; Cell Isolation; Cell Separation/Fractionation; Microscopy; Molecular/Chemical Probes.

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Extracellular Vesicles*
  • Gold
  • Mammals
  • Metal Nanoparticles*
  • Ultracentrifugation

Substances

  • Gold