Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

Summary

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

Abstract

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

Introduction

Extracellular vesicles (EVs) are small (0.03-2 µm) membrane-bound vesicles secreted by nearly all cell types¹. They are often referred to as "exosomes," "microvesicles," or "apoptotic bodies" depending on their mechanism of release and size². While it was initially thought that EVs were simply a means of eliminating waste from the cell to maintain homeostasis³, we now know that they can also participate in intercellular communication via transfer of molecular material - including DNA, RNA (mRNA, microRNA), lipids, and proteins^4,

Protocol

All work with these samples was performed in compliance with Institutional Animal Care and Use Committee and Institutional Review Board guidelines. A schematic overview of the NTA method is depicted in Figure 2.

Figure 2: Overview of NTA method using the particle tracking instrument. The sample is prepared and inserted into the instrument. The NTA software is opened, recording parameters are adjusted, and ....

Results

Before this demonstration, the calibration of the instrument was first tested to ensure the validity of the acquired data by measuring the size distribution of polystyrene bead standards. We tested the size distribution of 100 nm and 400 nm beads using the default recording parameters and the processing settings recommended in this protocol (Figure 8).

For the 100 nm polystyrene bead standard, a concentration of 4.205 x 10⁷ particles/mL was measured. Th.......

Discussion

Here, we demonstrate a protocol for NTA of EVs to measure the size distribution of a wide range of particle sizes simultaneously and measure total EV concentration in a polydisperse sample. In this study, mouse perigonadal adipose tissue and human plasma were used as the source of EVs. However, EVs isolated from other tissues or biological fluids such as serum, urine, saliva, breast milk, amniotic fluid, and cell culture supernatant may also be used for NTA. Measurements of polystyrene bead standards ensured that the ins.......

Materials

Name	Company	Catalog Number	Comments
1X dPBS	VWR	02-0119-1000	To dilute samples
100 nm bead standard	Thermo Scientific	3100A	To test ViewSizer 3000 calibration
400 nm bead standard	Thermo Scientific	3400A	To test ViewSizer 3000 calibration
Centrifugal Filter Unit	Amicon	UFC901024	To filter PBS diluent
Collection tubes, 2 mL	Qiagen	19201	For isolation of human plasma extracellular vesicles
Compressed air duster	DustOff	DPSJB-12	To clean cuvettes
Cuvette insert	HORIBA Scientific	-	Provided with purchase of ViewSizer 3000
Cuvette jig	HORIBA Scientific	-	To align magnetic stir bar while placing inserts inside cuvette; Provided with purchase of ViewSizer 3000
De-ionized water	VWR	02-0201-1000	To clean cuvettes
Desktop computer with monitor, keyboard, mouse, and all necessary cables	Dell	-	Provided with purchase of ViewSizer 3000
Ethanol (70-100%)	Millipore Sigma	-	To clean cuvettes
ExoQuick ULTRA	System Biosciences	EQULTRA-20A-1	For isolation of human plasma extracellular vesicles
Glass scintillation vials with lids	Thermo Scientific	B780020	To clean cuvettes
"Hook" tool	Excelta	-	Provided with purchase of ViewSizer 3000
Lint-free microfiber cloth	Texwipe	TX629	To clean cuvettes and cover work surface
Microcentrifuge tubes, 2 mL	Eppendorf	22363344	For isolation of human plasma extracellular vesicles
Stir bar	Sp Scienceware	F37119-0005
Suprasil Quartz cuvette with cap	Agilent Technologies	AG1000-0544	Initially provided with purchase of ViewSizer 3000
ViewSizer 3000	HORIBA Scientific	-	Nanoparticle tracking instrument