Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Summary

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Abstract

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

Introduction

Cells secrete extracellular vesicles (EV) of different sizes including microvesicles (MV) and exosomes (Exo). The latter can be distinguished from MV by both size and the subcellular compartment of origin. MV (200–1,000 nm in size) are released from parent cells by shedding from the plasma membrane. Conversely, Exo (30–150 nm) originate from endosomal membranes and are released into the extracellular space when the multivesicular bodies (MVB) fuse with the cell membrane^1,2.

EV are increasingly used as diagnostic biomarkers as well as, potentially, therapeutic tools in ma....

Protocol

1. Collection and Processing of Conditioned Media

1. Coat 55 cm² Petri dishes with 0.02% porcine skin gelatin in PBS.
2. Plate CPC (8,000/cm²) in pre-coated dishes with 7 mL of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% FBS (Fetal Bovine Serum) and 1% Penicillin/Streptomycin (P/S).
   NOTE: The term “CPC” refers to human explant derived cells that have been described elsewhere¹⁴. CM can be collected from different cell types cultured in specific culture conditions. Wear gloves and work under a biological hood.
3. Once cells reach about 80....

Results

Total number of particles for single staining

Since a single bead can bind more than one particle, we tested different conditions to set the smallest amount of total EV (single antibody per tube) to reach the early exponential phase of MFI curve. A fixed concentration of antibody was used while the total number of particles ranged from 5 x 10⁵ to 2.5 x 10⁸. As shown in .......

Discussion

Conventional FC technique remains the most straightforward analytic method to characterize markers expressed onto the surface of EV. In this regard, selecting the most appropriate protocol is crucial to obtain useful information on individual particle fractions of interest by avoiding limitations due to instrument sensitivity. We described a method using magnetic particles coupled with antibodies that recognize Exo and small EV surface antigens which are suitable for downstream FC application. We validated the method usi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IMDM	Gibco	12440061
Amicon Ultra-15, PLHK Ultracel-PL Membran, 100 kDa	Millipore	UFC910024
CytoFlex, Flow Cytometer Platform	Beckman Coulter	CytoFlex
DMEM, high glucose, HEPES, no phenol red	Gibco	21063045
Dulbecco's PBS (PBS) Ca- and Mg-free	Lonza	BE17-512F
ExoCap CD63 Capture Kit	JSR Life Sciences	Ex-C63-SP
ExoCap CD81 Capture Kit	JSR Life Sciences	Ex-C81-SP
ExoCap CD9 Capture Kit	JSR Life Sciences	Ex-C9-SP
Exosome-Depleted FBS	Thermofisher	A2720801
Exosome-depleted FBS Media Supplement	SBI	EXO-FBS-250A-1
FBS-Fetal Bovine Serum	Gibco	10270106
FITC anti-human CD9 Antibody	Biolegend	312104           RRID: AB_2075894
Flow Cytometer analysis software	Beckman Coulter	Kaluza
NanoSight LM10	Malvern	NanoSight LM10
NanoSight Software	Malvern	NTA 2.3
Optima Max-XP	Beckman Coulter	393315
PE anti-human CD63 Antibody	Biolegend	353004           RRID:AB_10897809
PE anti-human CD81 (TAPA-1) Antibody	Biolegend	349505           RRID:AB_10642024
Penicillin-Streptomycin	Gibco	15140122
Thermomixer C	Eppendorf	5382 000 015
TLA-110	Beckman Coulter	TLA-110 rotors