Protocol for differential centrifugation-based separation and characterization of apoptotic vesicles derived from human mesenchymal stem cells

Da Chen; Zixuan Zhao; Kai Zhang; Fang Jin; Chenxi Zheng; Yan Jin

doi:10.1016/j.xpro.2022.101695

Protocol for differential centrifugation-based separation and characterization of apoptotic vesicles derived from human mesenchymal stem cells

STAR Protoc. 2022 Dec 16;3(4):101695. doi: 10.1016/j.xpro.2022.101695. Epub 2022 Sep 20.

Authors

Da Chen¹, Zixuan Zhao¹, Kai Zhang², Fang Jin¹, Chenxi Zheng³, Yan Jin⁴

Affiliations

¹ State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China; Department of Orthodontics, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
² State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China; Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
³ State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Electronic address: chenxi199202@163.com.
⁴ State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Electronic address: yanjin@fmmu.edu.cn.

Abstract

Apoptotic vesicles (apoVs) are specific extracellular vesicles generated during apoptosis and play important roles in multiple physiological and pathophysiological settings. Here, we present a protocol using differential centrifugation to separate apoVs from human mesenchymal stem cells (MSCs) after induction of apoptosis. We describe how to characterize apoV size and morphology by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), and determination of specific biomarker expression by immunoblotting. Our protocol will be useful for preparing apoVs for further functional analysis. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021).

Keywords: Biotechnology and bioengineering; Cell biology; Cell separation/fractionation; Health sciences; Microscopy; Stem cells.

Publication types

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

MeSH terms

Apoptosis
Centrifugation
Extracellular Vesicles* / metabolism
Humans
Immunoblotting
Mesenchymal Stem Cells*