Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking...

Area of Science:

• Biotechnology
• Nanotechnology
• Cell Biology

Background:

• Extracellular vesicles (EVs) are crucial for intercellular communication, necessitating robust characterization methods.
• Accurate EV phenotyping is vital for understanding their biological roles and for therapeutic applications.
• Current methods for EV surface marker identification are often indirect or lack single-vesicle resolution.

Purpose of the Study:

• To develop and validate a rigorous protocol for indirect fluorescent immunolabeling of single extracellular vesicles (EVs).
• To simultaneously assess EV concentration, particle size distribution, and surface immunophenotype using nanoparticle tracking analysis (NTA).
• To establish a reliable method for quantifying EV surface epitope expression.

Main Methods:

• Isolation of EVs from canine and human cell cultures.
• Indirect fluorescent immunolabeling of EVs using quantum dots (Qds).
• Characterization via NTA in both light scatter and fluorescent modes, complemented by transmission electron microscopy and Western blotting.
• Application of bead-assisted depletion and size-exclusion chromatography to enhance labeling specificity.

Main Results:

• Reproducible detection of individual fluorescently labeled EVs was achieved using NTA.
• A method for evaluating immunolabeling success was proposed based on paired particle detection in NTA.
• Bead-assisted depletion and size-exclusion chromatography improved the specificity of quantum dot labeling.
• The developed method accurately estimated the fraction of EVs expressing specific epitopes.

Conclusions:

• The described indirect immunolabeling and NTA method provides an improved approach for EV characterization.
• This technique enables simultaneous assessment of EV concentration, size distribution, and surface immunophenotype at the single-vesicle level.
• This advancement facilitates more accurate comparisons of sample phenotypes and enriches downstream EV investigations.