Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization

Published: April 30, 2024

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) 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. 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. At the end of the procedure, after adding 10 µl of the quenching buffer, transfer MNPs t…

Disclosures

The authors have nothing to disclose.

Materials

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

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Cite This Article
Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization. J. Vis. Exp. (Pending Publication), e22120, doi: (2024).

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