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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200122	1/1	Homo sapiens	293 F	Filtration	Paganini, Carolina	2021	75%
Study summary Full title Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence-Based Microfluidic Diffusion Sizing All authors Carolina Paganini, Britta Hettich, Marie R G Kopp, Adam Eördögh, Umberto Capasso Palmiero, Giorgia Adamo, Nicolas Touzet, Mauro Manno, Antonella Bongiovanni, Pablo Rivera-Fuentes, Jean-Christophe Leroux, Paolo Arosio Journal Advanced Healthcare Materials Abstract Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variet (show more...)Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development. (hide) EV-METRIC 75% (96th 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 Cell culture supernatant 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 Filtration Protein markers EV: TSG101/ Alix/ CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 293 F EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count 2.20E+08 Separation Method Filtration steps > 0.45 µm, 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 CD63/ TSG101/ Alix/ CD81 Not detected contaminants Calnexin Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 23.2+/-6.6 NTA Report type Mean Reported size (nm) 133.6+/-1.2 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.30E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV200122
species	Homo sapiens
sample type	Cell culture
cell type	293 F
condition	Cell culture
separation protocol	Filtration
Exp. nr.	1
EV-METRIC %	75