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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180002	2/2	Canis familiaris	Mesenchymal stromal cells of placental origin	DG (d)(U)C	Thane KE	2019	88%
Study summary Full title Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking analysis. All authors Thane KE, Davis AM, Hoffman AM. Journal Sci Rep Abstract Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve (show more...)Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration. (hide) EV-METRIC 88% (98th 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 DG (d)(U)C Adj. k-factor 122.2 (pelleting) Protein markers EV: TSG101/ CD81/ CD9 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Canis familiaris Sample Type Cell culture supernatant EV-producing cells Mesenchymal stromal cells of placental origin EV-harvesting Medium Serum free medium 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 Type 70.1Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 122.2 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.1 Highest density fraction 0.4 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 120 Fraction volume (mL) 0.625 Fraction processing Ultrafiltration Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes EM EM-type Immune-EM/ Atomic force-EM EM protein CD9 Image type Close-up, Wide-field

	EV180002	1/2	Homo sapiens	HEK293	(d)(U)C	Thane KE	2019	14%
Study summary Full title Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking analysis. All authors Thane KE, Davis AM, Hoffman AM. Journal Sci Rep Abstract Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve (show more...)Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration. (hide) EV-METRIC 14% (44th 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 (d)(U)C Adj. k-factor 122.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium Serum free medium 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 Type 70.1Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 122.2 Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes

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EV-TRACK ID	EV180002
species	Canis familiaris	Homo sapiens
sample type	Cell culture	Cell culture
cell type	Mesenchymal stromal cells of placental origin	HEK293
condition	Control condition	Control condition
separation protocol	DG (d)(U)C	(d)(U)C
Exp. nr.	2	1
EV-METRIC %	88	14