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