Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Summary

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

Abstract

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

Introduction

Extracellular vesicles (EVs) are nanometer-sized membrane vesicles of cellular origin that can be isolated from numerous biological fluids, including blood, breast milk, saliva, urine, bile, pancreatic juice, and cerebrospinal and peritoneal fluids. Derivation of EVs occurs via three main mechanisms: apoptosis, release via fusion of multivesicular bodies with the plasma membrane, and blebbing of the plasma membrane¹. Evidence for EV transfer of donor cell components to neighboring or distant cells and tissues suggests these membrane enclosed packages may play important roles in paracrine as well as long distance or endocrine s....

Protocol

Serum samples were collected from mice according to an approved Institutional Animal Care and Use Committees (IACUC) at the University of Kansas Medical Center (KUMC) protocol. Use of these biological samples in these experiments was also approved by KUMC.

1. Sample preparation (Day 1)

1. Determine EV concentration using nanoparticle tracking or an equivalent technique.
2. Dilute the sample with the incubation solution to a concentration of 5 x 10⁷-5 x 10⁸ EVs/mL; a minimum of 50 µL is needed.
   NOTE: For samples in which the EV concentration is unknown, total protein at 1 µg/mL can ....

Results

Figure 7 (left panel) shows a three-color composite image of EVs derived from HEK293 conditioned media bound to the CD63 spot on the chip and stained for CD81, CD63, and CD9 in the following channels green, red, and blue, respectively. Figure 7 (top right panel) is a zoomed-in image that shows each of the captured EVs can display co-localization of one or more colors with varying intensities in each channel. The difference in staining of the captured EVs represe.......

Discussion

Current EV characterization methods largely rely on purified EVs, which is restricted by current experimental limitations of EV purification methods^{9,10,11,12,13}.Single-particle interferometric reflectance imaging (SP-IRIS) is an effective technology that can eliminate purification steps required for sample analysis and therefore save time and reduce costs tha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-cm sterile Petri dish	Fisher	FB0875712
15mL sterile tube	n/a	various
24-well cell culture plate, flat bottom	Fisher	08-772-1
Blocking Solution	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Chipfiles	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Chips	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Chuck	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Corning Easy Grip Disposable Polystyrene Sterile Bottles 250 ml	Fisher	09-761-4
Corning Easy Grip Disposable Polystyrene Sterile Bottles 500 ml	Fisher	09-761-10
Deionized (DI) water	Fisher	LC267404
EMS style tweezers with Carbon Fiber tips	Fisher	50-193-0842
ExoView Human Tetraspanin Kit	NanoView Biosciences	EV-TETRA-C	Capture for hCD81, hCD9, hCD63, IgG Control  + stains for hEV-A (hEV-CD63-647, hEV-CD81-555, hEV-CD9-488) 16 Chips per kit
ExoView R100 Imager	NanoView Biosciences	EV-R100	Interferometric microscope including high specification camera including 3 color fluorescence and label free sizing and counting extracellular vesicles
Fluorescently labled huma CD9 IgG antibody	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Fluorescently labled human CD63 IgG antibody	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Fluorescently labled human CD81 IgG antibody	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Incubation Solution	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Orbital shaker or microplate shaker with digital settings capable of shaking at 500 rpm	n/a	various
Plate Seal	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Solution A	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Solution B	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Solution C	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Solution D	NanoView Biosciences	EV-TETRA-C	Can be found in ExoView Human Tetraspanin Kit.
Square/flat tip tweezer	Fisher	50-239-62
Straight strong point Boley style tweezers	Fisher	16-100-124
Thermo Scientific Adhesive PCR Plate Seals	Fisher	AB-0558