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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200034	1/1	Homo sapiens	A549	(d)(U)C DG Filtration	Xiaohui Chen	2020	56%
Study summary Full title High-Fidelity Determination and Tracing of Small Extracellular Vesicle Cargoes All authors Xiaohui Chen, Mei Jia, Lianhua Liu, Xiaopei Qiu, Hong Zhang, Xingle Yu, Wei Gu, Guangchao Qing, Qingmei Li, Xiaolin Hu, Ruixuan Wang, Xianxian Zhao, Liangliang Zhang, Xianfeng Wang, Colm Durkan, Nan Wang, Guixue Wang, Yang Luo Journal Small Abstract Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for eluci (show more...)Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs' molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs' microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics. (hide) EV-METRIC 56% (90th 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 Control condition Focus vesicles extracellular vesicle 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 (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: TSG101/ CD9/ CD81/ CD63/ actin-beta non-EV: None Proteomics no EV density (g/ml) 1.15 Show all info Study aim New methodological development/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A549 EV-harvesting Medium Serum free medium Cell viability (%) 99 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) 120 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Bottom-up Rotor type Not specified Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Ultracentrifugation Pelleting: duration (min) 60 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101/ CD9/ CD81/ CD63/ actin-beta Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 104 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV200034
species	Homo sapiens
sample type	Cell culture
cell type	A549
condition	Control condition
separation protocol	dUC Density gradient Filtration
Exp. nr.	1
EV-METRIC %	56