Flow cytometric procedures for deep characterization of nanoparticles.

Area of Science:

• Biotechnology
• Nanotechnology
• Analytical Chemistry

Background:

• Accurate identification and quantification of nano-sized particles, such as extracellular vesicles (EVs) and viruses, are crucial in biomedical research.
• The small size of these particles presents significant challenges for precise examination and analysis.
• Flow cytometry is recognized as a highly sensitive, efficient, and non-destructive technique for studying EVs.

Purpose of the Study:

• To optimize flow cytometry protocols for the accurate identification, quantification, and purification of extracellular vesicles (EVs) and virus-like particles.
• To minimize artefacts and errors in nano-sized particle counting, particularly those arising from the swarming effect.
• To adapt conventional flow cytometry for non-standard experimental contexts and develop a reliable fluorescent-EVs sorting protocol.

Main Methods:

• Development and optimization of flow cytometry protocols for nano-sized particle analysis.
• Comparison of different threshold strategies to enhance result specificity and reduce counting errors.
• Identification of critical parameters for applying conventional flow cytometry beyond its typical use cases.
• Implementation of a fluorescent-EVs sorting protocol using a standard cell sorter.

Main Results:

• The optimized flow cytometry protocol effectively reduces artefacts and errors in nano-sized particle counting, addressing the swarming effect.
• Specific threshold strategies were validated to ensure high specificity in particle identification and quantification.
• Key parameters for utilizing conventional flow cytometry in diverse experimental settings were identified.
• A highly reliable protocol for fluorescent-EVs sorting was successfully developed and demonstrated.

Conclusions:

• Optimized flow cytometry offers a robust solution for the accurate analysis of nano-sized particles, including EVs and viruses.
• The developed protocols enhance the reliability of particle quantification, purification, and sorting, advancing nanomedicine and diagnostics.
• This work provides essential guidelines for researchers seeking to leverage flow cytometry for nano-particle studies in various contexts.