A compendium of single extracellular vesicle flow cytometry

Area of Science:

• Extracellular vesicle (EV) research
• Biophysical characterization
• Flow cytometry (FCM) applications

Background:

• Flow cytometry (FCM) is a powerful tool for analyzing single extracellular vesicles (EVs), but instrument design for larger cells creates detection challenges.
• EV signals often overlap with background noise, complicating analysis and reproducibility in EV-rich fluids.
• Existing frameworks like MIFlowCyt-EV lack sufficient background information for conducting robust EV FCM experiments.

Purpose of the Study:

• To provide comprehensive background information for designing and executing reproducible EV FCM experiments.
• To bridge the gap in understanding the technical nuances of EV detection via FCM.
• To enhance the interpretation, reporting, and reproducibility of EV FCM data.

Main Methods:

• Compiling background knowledge on EVs and their interaction with light.
• Detailing FCM hardware, experimental design, and preanalytical considerations.
• Explaining sample preparation, assay controls, instrument calibration, and data reporting.

Main Results:

• The compendium offers foundational knowledge crucial for overcoming EV FCM challenges.
• It covers essential aspects from sample handling to data analysis for EV characterization.
• The information presented is applicable to other small particle detection within the EV size range.

Conclusions:

• This resource is vital for researchers aiming to conduct reliable and reproducible extracellular vesicle flow cytometry studies.
• By addressing technical limitations, it facilitates more accurate EV characterization and data interpretation.
• The principles discussed can be extended to the FCM analysis of other nanoscale particles like viruses and lipoproteins.