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: Figure3 shows the results of two independent experiments in which Anx5-positive EVs were detected using either FS triggering (blue symbols) or FL triggering (red symbols), for dilution factors ranging from 4× to 128×. The concentration of Anx5-positive EVs in diluted PFP samples is observed to decrease linearly with the reciprocal dilution factor. In addition, the mean fluorescence intensity of EVs detected by FL triggering remains constant over the entire dilution range (Fig. S6). These results indicate the absence of coincidence effects on the detection of Anx5-positive EVs. Therefore, as the experiments reported here were performed with 10 ×-diluted PFP samples, we conclude that the 22 000 Anx5-positive EVs determined by FL triggering correspond to individual EVs.
Affiliation: Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN CNRS-University of Bordeaux-IPB, Pessac, France.