Abstract
Extracellular vesicles (EVs) range in size from 50 nm to 1 μm. Flow cytometry (FCM) is the most commonly used method for analyzing EVs; however, accurate characterization of EVs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques that are especially well-suited for analyzing EVs from a high volume of clinical samples. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-EV events. In addition, we show that lysed samples are a useful alternative to isotypes for setting gates to exclude background fluorescence. To reduce background, we developed an approach using filters to "wash" samples post-staining thus providing a faster alternative to ultracentrifugation and sucrose gradient fractionation. In conclusion, use of these optimized techniques enhances the accuracy and efficiency of EV detection using FCM.
Original language | English |
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Pages (from-to) | 1052-1063 |
Number of pages | 12 |
Journal | Cytometry Part A |
Volume | 87 |
Issue number | 11 |
DOIs | |
State | Published - Nov 2015 |
Keywords
- Antibody aggregates
- Extracellular vesicles
- Filtration
- Flow cytometry
- Microparticles