Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization

Abstract

Source: Arakelyan, A. et al., Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles. J. Vis. Exp. (2017)

This video demonstrates the immunocapture of extracellular vesicles using fluorescent-labeled capture antibody-magnetic nanoparticle conjugates. The process involves complex formation, staining with fluorescently labeled detection antibodies, magnetic separation, and flow cytometric analysis for antigenic characterization.

Protocol

1. Coupling of Magnetic Nanoparticles (MNPs)

1. Use commercial coupling procedure and reagents to couple MNPs with antibodies (Abs) (typically 1 mg). Iron oxide nanoparticles with carboxylic acid as reactive groups are used.
   1. If antibodies are in a volume higher than 0.5 ml, concentrate using a 100K concentrator. Spin at 2,000 x g for 3-5 min.
2. At the end of the procedure, after adding 10 µl of the quenching buffer, transfer MNPs to a 12 mm x 75 mm tube and add 3 ml wash/storage buffer. Place the tube in the center hole of a magnetic separator and leave overnight at 4 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Carboxyl Magnetic Iron Oxide Nanoparticles Conjugation Kits	Ocean Nanotech	ICK-15-005
Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-100 membrane	Millipore	UFC810024
5mL Round Bottom PP Test Tube, without Cap	Falcon	352002
DEPC treated water	Quality Biological	351-068-131
SuperMAG-01 magnetic separator	Ocean Nanotech	SuperMAG-01
Zenon R-Phycoerythrin Mouse IgG1 Labeling Kit	Thermo Fisher Scientific	Z25055
Nanosep Centrifugal Devices with Omega Membrane 100k	Pall Corp	OD100C34
EDTA 0.5M	Corning Cellgro	46-034-CI
Bovine Serum Albumin solution	Sigma-Aldrich	A9576-50ML
PBS	Gibco	10010-23
Paraformaldehyde, EM Grade, Purified	EMS	15710
123count eBeads	eBioscience	01-1234-42
CytoCount beads	Dako	S2366
AccuCheck count beads	Invitrogen	PCB100
Cytometer Setup & Tracking Beads Kit (CST)	BD Bioscience	642412
Equipment
µMACS column	Miltenyi Biotec	130-042-701
OctoMACS Separator magnet	Miltenyi Biotec	130-042-108
Nanodrop	ThermoFisher	none
NanoSight NS300	Malvern	none
BD LSRII flow cytometer	BD Bioscience	none
Software
Flowjo software	Flowjo
BD FACSDiva software	BD