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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180010	2/2	Homo sapiens	Serum	DG (d)(U)C	Busatto, Sara	2018	88%
Study summary Full title Uptake Profiles of Human Serum Exosomes by Murine and Human Tumor Cells through Combined Use of Colloidal Nanoplasmonics and Flow Cytofluorimetric Analysis All authors Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, Paolo Bergese Journal Anal Chem Abstract Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capi (show more...)Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth. (hide) EV-METRIC 88% (99th 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 Serum Sample origin Control condition Focus vesicles exosome 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 89.2 (pelleting) Protein markers EV: Alix/ CD81/ ANXA11/ CD63 non-EV: GM130/ APOA1 Proteomics no Show all info Study aim Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 TLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 89.20 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.15 Highest density fraction 0.6 Sample volume (mL) 0.8 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.4 Fraction processing Centrifugation Pelleting: volume per fraction 0.4 Pelleting: duration (min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, CD63, CD81, ANXA11 Not detected contaminants GM130, APOA1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 70 EM EM-type Atomic force-EM Image type Wide-field Report size (nm) 30-150 Other particle analysis name(2) Colorimetric nanoplasmonic assay (CONAN) EV-concentration Yes Particle yield 5.30E+12

	EV180010	1/2	Homo sapiens	Serum	(d)(U)C qEV	Busatto, Sara	2018	44%
Study summary Full title Uptake Profiles of Human Serum Exosomes by Murine and Human Tumor Cells through Combined Use of Colloidal Nanoplasmonics and Flow Cytofluorimetric Analysis All authors Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, Paolo Bergese Journal Anal Chem Abstract Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capi (show more...)Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth. (hide) EV-METRIC 44% (86th 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 Serum Sample origin Control condition Focus vesicles exosome 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 qEV Adj. k-factor 89.2 (pelleting) Protein markers EV: ANXA11/ CD63 non-EV: GM130/ APOA1 Proteomics no Show all info Study aim Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 TLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 89.20 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, ANXA11 Not detected contaminants GM130, APOA1 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force-EM Image type Wide-field Report size (nm) 30-150

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EV-TRACK ID	EV180010
species	Homo sapiens
sample type	Serum
condition	Control condition
separation protocol	DG (d)(U)C	(d)(U)C qEV
Exp. nr.	2	1
EV-METRIC %	88	44