Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Summary

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Abstract

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal–to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Introduction

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for process....

Protocol

NOTE: The following protocols have been performed in compliance with all institutional, national and international guidelines for human welfare. All human subject samples were tested under an institutional review board (IRB)-approved protocol and with informed consent of the subjects.

1. METHOD A: Individual Detection Method

1.1) Processing of Blood Sample/Isolation of EVs

1. Draw blood from donor/patient into two 10 ml glass tubes containing 1.5 ml of ACD-Solution A or other suitable anticoagulant and process immediately (within 30 min max) using the following 2-step differential centrifugation protocol.

Results

Figure 1 outlines the overall processing scheme for the isolation and detection of EVs using either the bead-based method or individual detection method. Individual detection of EVs using FCM works well for analyzing larger EVs but most cytometers are not capable of individually detecting particles as small as exosomes. A bead-based approach allows small EVs to be detected, however, there are drawbacks associated with using this method, as outlined in Table 1. Generally, isolation of EVs.......

Discussion

Two different protocols for the isolation, treatment and analysis of EVs were presented, using either an individual detection or bead-based approach. Selecting the most appropriate method to use is not always straightforward and requires an understanding of the sample being tested as well as the individual subpopulations of interest. Furthermore, the sensitivity of the cytometer used for acquisition must be considered when choosing the most appropriate method. Oftentimes there is no single best protocol to use, rather, a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LSR II benchtop flow cytometer	BD Biosciences		3-laser (20 mW Coherent Sapphire 488 nm blue, 25 mW Coherent Vioflame 405 nm violet, and 17 mW JDS Uniphase HeNe 633 nm red)
FACS Diva software	BD Biosciences		PC version 6.0
FlowJo software	Treestar US		Mac version 9.6.1 or PC version 7.6.5
Sphero Rainbow fluorescent particles	BD Biosciences	556298	used to adjust all channel voltages to maintain fluorescence intensity consistency
Ultra Rainbow fluorescent particles	Spherotech	URFP-10-5	used in addition to Megamix-Plus SSC beads to ensure EV gating consistency from batch to batch
Megmix-Plus SSC beads	Biocytex	7803	used to adjust FSC and SSC  voltages to maintain  consistency  between runs. Can also used to monitor flow rate and ajust flow rate dial in order to ensure that same flow rate is used in all runs
AbC Anti-Mouse Bead Kit	Life Technologies	A-10344	used for compensation controls & negative AbC beads used for beads-based method
Nonidet P-40 Alternative (NP-40) (CAS 9016-45-9)	Santa Cruz	sc-281108	used in the individual detection method only to lyse samples after initial reading for use as negative controls. Stock may be diluted to 1:10 in PBS and stored in fridge for up to 1 month.
BD TruCOUNT Tubes	BD Biosciences	340334	used whenver absolute EV concentrations are needed
Ultrafree-MC, GV 0.22 µm Centrifugal Filter Units	Millipore	UFC30GVNB	used to post-stain wash Evs and/or fractionate EVs based on size
Vacutainer glass whole blood tubes ACD-A	BD Biosciences	364606
Facs tubes 12x75 polystrene	BD Biosciences	352058
50 ml Reservoirs individually wrapped	Phenix	RR-50-1s
Green-Pak pipet tips - 10 µl	Rainin	GP-L10S
Green-Pak pipet tips -200 µl	Rainin	GP-L250S
Green -Pak pipet tips - 1,000 µl	Rainin	GP-L1000S
Stable Stack L300 tips presterilized	Rainin	SS-L300S
Pipet-Lite XLS 8 Channel LTS Adjustable Spacer	Rainin	LA8-300XLS
96 well tissue culture plates	E&K Scientific	EK-20180
RPMI 1640 Media (without Hepes)	UCSF Cell Culture Facility	CCFAE001	media used for bead-based detection method
Dulbeccos PBS D-PBS, CaMg-free, 0.2 µm filtered	UCSF Cell Culture Facility	CCFAL003
Ultracentrifuge Tube, Thinwall, Ultra-Clear	BECKMAN COULTER INC	344058
PANEL I
CD3 PerCP-Cy5.5	Biolegend	344808	2 µl
CD14 APC-Cy7	Biolegend	301820	2 µl
CD16 V450	BD Biosciences	560474	2 µl
CD28 FITC	biolegend	302906	2 µl
CD152 APC	BD Biosciences	555855	2 µl
CD19 A700	Biolegend	302226	2 µl
PANEL II
CD41a PerCP-Cy5.5	BD Biosciences	340930	2 µl
CD62L APC	Biolegend	304810	2 µl
CD108 PE	BD Biosciences	552830	2 µl
CD235a FITC	biolegend	349104	2 µl
PANEL III
CD11b PE-Cy7	Biolegend	301322	2 µl
CD62p APC	Biolegend	304910	2 µl
CD66b PE	Biolegend	305106	2 µl
CD15 FITC	exalpha	X1496M	5 µl
CD9 PE	Biolegend	555372
CD63 APC	Biolegend	353008
APC-Cy7 Ms IgG2a, κ	Biolegend	400230