A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles.
Bottom Line: We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering.By fluorescence triggering, concentrations of 22 000-30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers.Results from EM suggest that EVs down to 100-150 nm diameter are detected by fluorescence triggering.
Affiliation: Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN CNRS-University of Bordeaux-IPB, Pessac, France.Show MeSH
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Mentions: PFP samples were first analyzed by a conventional FCM approach, in which the trigger was set on the forward scatter (FS) parameter and the detection was limited to events labeled by Anx5-F* (Fig.1A,B). The concentration of Anx5-positive EVs detected by this approach was 457 ± 178 (n = 10) per μL pure PFP. This value is consistent with previous reports on EVs from normal PFP [20,23,28,30]. Displayed on a FS vs. side scatter (SS) color dot plot, most of the Anx5-positive EVs are superposed with the background from the buffer (Fig.1A,B). On an FL1 vs. SS plot, two populations of Anx5-positive EVs can be distinguished. About 30% of them (134 ± 91 per μL) form a distinct cluster of high fluorescence intensity events (colored blue in Fig.1B). As previously reported , these objects, which are characterized by a high fluorescence intensity and scattering properties similar to 500-nm polymer particles (Fig.1B left), correspond to empty erythrocytes and are called hereafter erythrocyte ghosts. The rest of the Anx5-positive EVs are colored red in Fig.1B.
Affiliation: Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN CNRS-University of Bordeaux-IPB, Pessac, France.