ABSTRACT
Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The study of EV secretion and EV characterization remains challenging due to the small size of these particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV separation techniques. We developed a method for in-cell EV labeling with fluorescent lipids (DiI), followed by DiI-labelled EV characterization in the conditioned medium by imaging flow cytometry (IFC). Direct IFC analysis of EVs in the conditioned medium, after removal of apoptotic bodies and cellular debris, significantly reduces sample processing and loss compared to established methods for EV separation, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by ultracentrifugation. In conclusion, our optimized protocol for EV labeling and analysis reduces EV sample processing and loss, and is well suited for cell biology studies that focus on modulation of EV secretion by cells in culture.
Competing Interest Statement
The authors have declared no competing interest.
- Abbreviations
- Baf
- Bafilomycin A1;
- BCA
- Bicinchoninic acid;
- EVs
- Extracellular vesicles;
- CCM
- cleared conditioned medium;
- FBS
- Fetal bovine serum;
- IFC
- Image Flow Cytometry;
- LC3
- Microtubule-associated protein light chain 3;
- NTA
- Nanoparticle tracking analysis;
- PBS
- Phosphate buffered saline;
- PS
- Phosphatidylserine;
- SEC
- Size exclusion chromatography;
- TEM
- Transmission electron microscopy;
- UC
- Differential ultracentrifugation;
- UCM
- Unconditioned media.