High Purity Isolation and Sensitive Quantification of Extracellular Vesicles Using Affinity to TIM4

Takeshi Yoshida1, Takamasa Ishidome1, Rikinari Hanayama1

1 Department of Immunology, Kanazawa University Graduate School of Medical Sciences, Kanazawa
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.45
DOI:  10.1002/cpcb.32
Online Posting Date:  December, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Almost all types of cells secrete extracellular vesicles (EVs), including exosomes and microvesicles. EVs carry various proteins, lipids, mRNAs, and microRNAs, and may participate in many aspects of physiological and pathophysiological processes. Various studies are currently being conducted to develop therapeutic and diagnostic methods targeting or utilizing EVs. Therefore, developing ideal methods for isolating and quantifying EVs is an active area of research. EVs express phosphatidylserine on their outer lipid bilayer. This unit describes an affinity‐based method for isolating EVs using TIM4, which binds phosphatidylserine in a specific and calcium‐dependent manner. EVs captured by TIM4 can be easily released by addition of a chelating agent, or can be retained for quantification by ELISA or flow cytometry. These methods enable the isolation of highly purified EVs and the sensitive quantification of EVs, which will accelerate EV research beyond what has been achievable with conventional methods. © 2017 by John Wiley & Sons, Inc.

Keywords: exosomes; extracellular vesicles; microvesicles; phosphatidylserine; TIM4

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of 10K Supernatants
  • Alternate Protocol 1: Preparation of EV‐Free FBS by PEG Precipitation
  • Support Protocol 1: Buffer Exchange of the 10K Sup
  • Basic Protocol 2: Preparation of TIM4 Affinity Beads
  • Basic Protocol 3: Isolation of Highly Purified EVs with TIM4 Affinity Beads
  • Support Protocol 2: Buffer Exchange of Isolated EVs Before a Physiological Assay
  • Basic Protocol 4: Quantification of Isolated EVs by Nanoparticle Tracking Analysis
  • Alternate Protocol 2: Quantification of Total Protein in Isolated EVs Using the BCA Assay
  • Alternate Protocol 3: Quantification of Surface Marker Proteins on Isolated EVs by Immunoblot Analysis
  • Basic Protocol 5: Quantification of EVs by TIM4 Affinity ELISA
  • Basic Protocol 6: Analysis of EV Surface Proteins by TIM4 Affinity Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of 10K Supernatants

  Materials
  • Fetal bovine serum (FBS)
  • Basic culture medium (e.g., MEM‐α, RPMI‐1640, DMEM)
  • Cells or biological fluids of interest
  • Ultracentrifuge with swinging‐bucket rotor and tubes (e.g., Beckman Optima X series with SW41Ti rotor)
  • 0.22‐μm hydrophilic PVDF membrane filter units
  • 60‐, 100‐, or 150‐mm culture dishes
  • 15‐ml polypropylene tubes

Alternate Protocol 1: Preparation of EV‐Free FBS by PEG Precipitation

  Materials
  • TIM4‐Fc protein (Adipogen or Wako)
  • EZ‐Link maleimide‐PEG11‐biotin (Thermo Fisher Scientific)
  • TBS (see recipe)
  • Dynabeads MyOne Streptavidin C1 Magnetic Beads (Thermo Fisher Scientific)
  • Preservative buffer (see recipe)
  • 1.5‐ml low‐adsorption microcentrifuge tubes
  • Ultrafiltration column with 10‐kDa MWCO (e.g., Millipore UFC5010)
  • Magnetic stand (e.g., Promega, Thermo Fisher Scientific, Bio‐Rad)
  • Rotator

Support Protocol 1: Buffer Exchange of the 10K Sup

  Materials
  • TIM4 affinity beads (see protocol 4)
  • 10K supernatant or 10K pellet (see protocol 1 or protocol 3)
  • 500× binding enhancer (see recipe)
  • Elution buffer (see recipe)
  • Wash buffer (see recipe)
  • Preservative buffer (see recipe)
  • Magnetic stand (e.g., Promega, Thermo Fisher Scientific, Bio‐Rad)
  • Rotator
  • 1.5‐ml low‐adsorption microcentrifuge tubes

Basic Protocol 2: Preparation of TIM4 Affinity Beads

  Materials
  • Affinity‐purified EVs (see protocol 5)
  • PBS (see recipe) or other saline, buffer, or medium without FBS
  • Ultrafiltration column with 10‐kDa MWCO (e.g., Millipore UFC5010)
  • 1.5‐ml microcentrifuge tubes
  • Dialysis membrane with 3.5‐kDa MWCO (Spectrum Labs)
  • Tubing closures (Spectrum Labs)
  • 300‐ml beaker

Basic Protocol 3: Isolation of Highly Purified EVs with TIM4 Affinity Beads

  Materials
  • Affinity‐purified EVs (see protocol 5 or protocol 6)
  • PBS (see recipe)
  • 1.5‐ml tubes
  • Nanoparticle tracking analyzer (NanoSIGHT LM10, Malvern Instruments)

Support Protocol 2: Buffer Exchange of Isolated EVs Before a Physiological Assay

  Materials
  • Affinity‐purified EVs (see protocol 5 or protocol 6)
  • Bovine serum albumin (BSA) standard
  • PBS (see recipe)
  • Ultrafiltration column with 10‐kDa MWCO (e.g., Millipore, GE)
  • 1.5‐ml microcentrifuge tubes
  • 96‐well assay plate (e.g., Thermo Fisher Scientific, Greiner, Iwaki)
  • Pierce BCA protein assay kit (e.g., Thermo Fisher Scientific, Takara)
  • Incubator set at 37°C
  • Plate reader with filter for wavelength between 540 and 590 nm (e.g., Bio‐Rad, Perkin Elmer)

Basic Protocol 4: Quantification of Isolated EVs by Nanoparticle Tracking Analysis

  Materials
  • Affinity‐purified EVs (see protocol 5 or protocol 6)
  • 5× SDS sample buffer (see recipe)
  • Additional reagents and equipment for SDS‐PAGE and immunoblotting (units 6.1 & 6.2; Gallagher, 3.45; Ni, Xu, & Gallagher, 3.45)

Alternate Protocol 2: Quantification of Total Protein in Isolated EVs Using the BCA Assay

  Materials
  • TIM4‐Fc protein (Adipogen or Wako)
  • Coating buffer (see recipe)
  • TBS‐T (see recipe)
  • 1% (w/v) bovine serum albumin (BSA) in TBS‐T
  • Wash buffer (see recipe)
  • 10K supernatant and/or pellet (see protocol 1)
  • 10× binding enhancer (see recipe)
  • Primary antibody (e.g., anti‐human CD9, anti‐human CD63, anti‐human CD81)
  • HRP‐conjugated secondary antibody (e.g., Millipore, Jackson)
  • TMB reagent (3,3′,5,5′‐tetramethylbenzidine; e.g., Thermo Fisher Scientific, Abcam, Nacalai)
  • 1 M H 2SO 4
  • 96‐well ELISA plate (e.g., Thermo Fisher Scientific, Greiner, Iwaki)
  • Plate reader with filter for 450 nm wavelength (e.g., Bio‐Rad, Perkin Elmer)

Alternate Protocol 3: Quantification of Surface Marker Proteins on Isolated EVs by Immunoblot Analysis

  Materials
  • Streptavidin‐coated magnetic beads (e.g., Exosome‐Streptavidin Isolation/Detection, Thermo Fisher Scientific)
  • Preservative buffer (see recipe)
  • Biotinylated TIM4‐Fc protein (see protocol 4)
  • 10K supernatant (see protocol 1)
  • 10× binding enhancer (see recipe)
  • Fluorescent anti‐EV marker antibody (e.g., FITC‐conjugated anti‐CD63)
  • 1.5‐ml low‐adsorption microcentrifuge tube
  • Magnetic stand (e.g., Promega, Thermo Fisher Scientific, Bio‐Rad)
  • Rotator (e.g., Taitec)
  • Flow cytometer
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Becker, A., Thakur, B. K., Weiss, J. M., Kim, H. S., Peinado, H., & Lyden, D. (2016). Extracellular vesicles in cancer: Cell‐to‐cell mediators of metastasis. Cancer Cell, 30, 836–848. doi: 10.1016/j.ccell.2016.10.009.
  Clayton, A., Court, J., Navabi, H., Adams, M., Mason, M. D., Hobot, J. A., … Jasani, B. (2001). Analysis of antigen presenting cell derived exosomes, based on immuno‐magnetic isolation and flow cytometry. Journal of Immunological Methods, 247, 163–174. doi: 10.1016/S0022‐1759(00)00321‐5.
  Colombo, M., Raposo, G., & Thery, C. (2014). Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annual Review of Cell and Developmental Biology, 30, 255–289. doi: 10.1146/annurev‐cellbio‐101512‐122326.
  Gallagher, S. R. (2007). One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Cell Biology, 37, 6.1.1–6.1.38.
  Kramer‐Albers, E. M., & Hill, A. F. (2016). Extracellular vesicles: Interneural shuttles of complex messages. Current Opinion in Neurobiology, 39, 101–107. doi: 10.1016/j.conb.2016.04.016.
  Miyanishi, M., Tada, K., Koike, M., Uchiyama, Y., Kitamura, T., & Nagata, S. (2007). Identification of Tim4 as a phosphatidylserine receptor. Nature, 450, 435–439. doi: 10.1038/nature06307.
  Nakai, W., Yoshida, T., Diez, D., Miyatake, Y., Nishibu, T., Imawaka, N., … Hanayama, R. (2016). A novel affinity‐based method for the isolation of highly purified extracellular vesicles. Scientific Reports, 6, 33935. doi: 10.1038/srep33935.
  Ni, D., Xu, P., & Gallagher, S. (2017). Immunoblotting and immunodetection. Current Protocols in Cell Biology, 74, 6.2.1‐6.2.37. doi: 10.1002/cpcb.18.
  Rider, M. A., Hurwitz, S. N., & Meckes, D. G., Jr. (2016). ExtraPEG: A polyethylene glycol‐based method for enrichment of extracellular vesicles. Scientific Reports, 6, 23978. doi: 10.1038/srep23978.
  Robbins, P. D., & Morelli, A. E. (2014). Regulation of immune responses by extracellular vesicles. Nature Reviews Immunology, 14, 195–208. doi: 10.1038/nri3622.
  Thery, C., Amigorena, S., Raposo, G., & Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protocols in Cell Biology, 30, 3.22.1‐3.22.29. doi: 10.1002/0471143030.cb0322s30.
  Tkach, M., & Thery, C. (2016). Communication by extracellular vesicles: Where we are and where we need to go. Cell, 164, 1226–1232. doi: 10.1016/j.cell.2016.01.043.
  Trajkovic, K., Hsu, C., Chiantia, S., Rajendran, L., Wenzel, D., Wieland, F., … Simons, M. (2008). Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science, 319, 1244–1247. doi: 10.1126/science.1153124.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library