Ultracentrifugal separation, characterization, and functional study of extracellular vesicles derived from serum-free cell culture

Hooi Ting Hu; Tamako Nishimura; Shiro Suetsugu

doi:10.1016/j.xpro.2021.100625

Ultracentrifugal separation, characterization, and functional study of extracellular vesicles derived from serum-free cell culture

STAR Protoc. 2021 Jun 23;2(3):100625. doi: 10.1016/j.xpro.2021.100625. eCollection 2021 Sep 17.

Authors

Hooi Ting Hu¹, Tamako Nishimura¹, Shiro Suetsugu^{1

2

3}

Affiliations

¹ Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma 630-0192, Japan.
² Data Science Center, Nara Institute of Science and Technology, Ikoma 630-0192, Japan.
³ Center for Digital Green-innovation, Nara Institute of Science and Technology, Ikoma 630-0192, Japan.

Abstract

Extracellular vesicles (EVs) play important roles in extracellular trafficking and signaling. Here, we separate EVs by differential centrifugation. EVs separated by this approach are called large EVs (l-EVs) and small EVs (s-EVs), reflecting particle size, which sediment based on different ultracentrifugation forces. The resulting EVs can be quantified and analyzed using nanoparticle tracking analysis, immunoblotting, and functional assays. This protocol was applied to a suspension cell line with high transfection efficiency adapted to a high-density, serum-free culture. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2021).

Keywords: Antibody; Biotechnology and bioengineering; Cell Membrane; Cell culture; Cell separation/fractionation; Molecular Biology; Protein Biochemistry; Signal Transduction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blotting, Western
Cell Movement
Culture Media, Serum-Free
Extracellular Vesicles / metabolism*
HEK293 Cells
Humans
Proteins / isolation & purification
Ultracentrifugation / methods*

Substances

Culture Media, Serum-Free
Proteins