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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140214	1/1	Homo sapiens	NAY	(d)(U)C DC Filtration	Salomon C	2014	33%
Study summary Full title Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration All authors Salomon C, Yee S, Scholz-Romero K, Kobayashi M, Vaswani K, Kvaskoff D, Illanes SE, Mitchell MD, Rice GE Journal Front Pharmacol Abstract BACKGROUND: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterin (show more...)BACKGROUND: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. METHODS: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software). RESULTS: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 10(8) ± 2.5 × 10(8) particles/10(6) cells) compared to JEG-3 (2.86 × 10(8) ± 0.78 × 10(8) particles/10(6) cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (-exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. CONCLUSION: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Adj. k-factor 221.9 (pelleting) Protein markers EV: CD63 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 75 Pelleting: rotor type SureSpin630 Pelleting: adjusted k-factor 221.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 ELISA Antibody details provided? No Detected EV-associated proteins CD63 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140214
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DC Filtration
Exp. nr.	1
EV-METRIC %	33