Abstract
Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration.
Highlights
Studies of extracellular vesicle (EV) biogenesis, disease relevance, and diagnostic and therapeutic potential have rapidly expanded over the last decade, prompting the advancement of rigorous methods to characterize the biophysical, phenotypic, and functional attributes of EV1
Nanoparticle tracking analysis (NTA)-FL using Quantum dots (Qd) has been plagued with several additional obstacles, including diminished reactivity of antibodies when directly conjugated with Qd and the absence of effective protocols to separate Qd from Qd-labeled extracellular vesicles (EV) during indirect immunolabeling protocols
We demonstrate that a combination of indirect immunolabeling utilizing Qd coupled with size-exclusion chromatography offers a robust method for exploiting NTA-FL for this purpose
Summary
Studies of extracellular vesicle (EV) biogenesis, disease relevance, and diagnostic and therapeutic potential have rapidly expanded over the last decade, prompting the advancement of rigorous methods to characterize the biophysical, phenotypic, and functional attributes of EV1. There are few reports demonstrating rigorous assessment of NTA-FL, and those which exist in the literature have relied upon immunolabeling non-canonical EV protein targets[30,31,32] or non-specific labeling[33]. These reports demonstrate the potential of this application and due to the instability of many fluorophores has led to the use of quantum dots for application with NTA-FL. The purpose of this study was to test a method for indirect fluorescent immunolabeling of single EV for NTA-FL that overcomes these shortfalls, facilitating single–vesicle immunolabeling and detection concurrent with particle size distribution and concentration. We demonstrate that a combination of indirect immunolabeling utilizing Qd coupled with size-exclusion chromatography offers a robust method for exploiting NTA-FL for this purpose
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
