H a heterogeneous morphology, whereas antibody immunogold labelling confirmed the presence of LEL tetraspanins on the surface of niosomes. Ultimately, using high-resolution flow cytometry, expression of recombinant tetraspanins was further confirmed in the single niosome-level. Summary/conclusion: We here describe the production of tetraspanindecorated nanovesicles. Using many isolation and detection tactics, we show that these nanovesicles have equivalent biophysical properties to EVs and are suited for antibody-staining approaches, producing these bioengineered nanovesicles an efficient normal and reference material for different EV-detection strategies. Funding: Grants from Fundaci Ram Areces and Ministerio de Econom y Competitividad (BFU2014-55478-R, REDIEX. SAF201571231-REDT). E.L. was supported by the ESF, GEIVEX Mobility and UAM STS fellowships.OT06.Isolation of microvesicles and exosomes by fluorescence-triggered FACS Celine Gounou1; Sisareuth Tan1; Nicolas Arraud2; Alain R. Brisson3 UMR-5248 CNRS – of Bordeaux, Pessac, France; 2Laboratoire de Cytom rie en Flux, H itaux Universitaires de Gen e, Geneva, Switzerland; 3University of Bordeaux, Pessac, FranceBackground: The isolation of extracellular vesicles (EV) Brutons Tyrosine Kinase (BTK) Proteins web constitutes a major challenge in the EV field, mainly on account of the heterogeneity of EV suspensions along with the difficulty of EV detection. We showed earlier that the detection of EVs was Carboxypeptidase D Proteins Source substantially enhanced by fluorescence-triggered flow cytometry (FL-FCM) as compared to standard lightscatter triggering (1).ISEV 2018 abstract bookThe objective of this study was two-fold: (1) to sort selected EV populations by FL FACS and (two) to evaluate the sorting efficiency on the two main EV populations, namely massive (one hundred nm to 1 ) microvesicles (MV) derived from plasma membranes and compact (5000 nm) exosomes derived from multivesicular bodies. Methods: MV had been obtained by hypotonic lysis of erythrocytes, when EX derived from reticulocytes had been obtained from sickle cell disease plasma. EV sorting was performed using a FACS-Aria-II (Becton Dickinson) utilizing PE-labelled anti-CD235a and anti-CD71 IgGs and Cy5-annexin5 (Anx5). In parallel to FCM, immuno-cryo-electron microscopy was utilized to image EV just before and right after sorting (two). Outcomes: Just before sorting, EV were very first characterized by FCM and immunocryoEM. Erythrocyte-derived MV consist of 10000 nm vesicles that expose each CD235a and phosphatidylserine, even though reticulocyte-derived EX consist of 5000 nm vesicles that express the transferrin receptor CD71 (three). The situations of sorting had been optimized for MV, working with FL-FCM based either on PE-CD235a-IgG or on Cy5-Anx5 signal, and for EX using FL-FCM on PE-CD71-IgG. The sorted MV and EX suspensions were re-analysed by FCM and by immuno-cryoEM. A sorting yield was calculated, equal to the ratio of EV concentrations detected by FL-FCM just before and just after sorting. Sorting yields of 200 were found for CD235a+ and PS + MV and 30 for CD71+ EX, respectively. Both EV suspensions had been of higher purity, as shown by immuno-cryoEM. Summary/conclusion: We show here that fluorescence-triggered FACS is really a strong and general technique for isolating EV, and for the very first time, that EV as little as exosomes is usually sorted with high efficiency using a typical FACS equipment. The isolation of chosen EV populations constitutes a significant step towards the determination of their omic composition plus the elucidation of their particular function. 1- Arraud et al. Cytometry A 2016 9:18.
