Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated...

Area of Science:

• Biotechnology
• Cell Biology
• Analytical Chemistry

Background:

• Flow cytometry is crucial for cell analysis but faces challenges with small extracellular vesicles (EVs) like exosomes due to their size and optical properties.
• Current methods, such as ultracentrifugation coupled with sucrose gradient flotation, are complex, require specialized operators, and involve extensive sample preparation.

Purpose of the Study:

• To develop a simplified, standardized, and user-friendly flow cytometry protocol for the quantification and characterization of exosomes and microvesicles.
• To enable routine EV analysis in non-specialized laboratories without the need for manual instrument adjustments.

Main Methods:

• Utilized a flow cytometer specifically designed for small particles, enabling analysis without prior hardware adjustments.
• Employed fluorescent labeling of EVs using protein- (CFSE) and lipid-specific (FM) dyes, compatible with antibody labeling.
• Implemented a bead-based standardization for improved control and reproducibility.

Main Results:

• Successfully quantified and characterized exosomes and microvesicles from cell culture media and human ascites samples.
• Demonstrated that double labeling with protein- and lipid-specific dyes effectively distinguishes EVs from contaminants like protein aggregates and dye micelles.
• Showcased the protocol's compatibility with antibody labeling for comprehensive EV characterization.

Conclusions:

• The presented methodology offers a fast, reliable, and accessible approach for routine quantification and characterization of EVs from diverse sources.
• The bead-based standardization and simplified workflow have the potential to enhance control and reproducibility in standard laboratory settings.