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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210135	1/2	Homo sapiens	Red blood cells	DG (d)(U)C UF	Stremersch, Stephan	2016	78%
Study summary Full title Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy All authors Stephan Stremersch, Monica Marro, Bat-El Pinchasik, Pieter Baatsen, An Hendrix, Stefaan C De Smedt, Pablo Loza-Alvarez, Andre G Skirtach, Koen Raemdonck, Kevin Braeckmans Journal Small Abstract Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention f (show more...)Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis. (hide) EV-METRIC 78% (97th 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 UF Protein markers EV: CD81/ HSP70/ CD63/ actin non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Red blood cells 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 Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 12.5% Highest density fraction 50% Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Top-down Rotor type SW 55 Ti Speed (g) 150000 Duration (min) 900 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 150000 Ultra filtration Cut-off size (kDa) 30 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ actin/ HSP70/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 170 Particle yield Not reported EM EM-type Cryo-EM Image type Wide-field EV concentration Not

	EV210135	2/2	Mus musculus	B16F10	DG (d)(U)C UF	Stremersch, Stephan	2016	78%
Study summary Full title Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy All authors Stephan Stremersch, Monica Marro, Bat-El Pinchasik, Pieter Baatsen, An Hendrix, Stefaan C De Smedt, Pablo Loza-Alvarez, Andre G Skirtach, Koen Raemdonck, Kevin Braeckmans Journal Small Abstract Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention f (show more...)Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis. (hide) EV-METRIC 78% (97th 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 UF Protein markers EV: CD81/ HSP70/ CD63/ actin non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods/New methodological development Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16F10 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;Ultrafiltration 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 Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 12.5% Highest density fraction 50% Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Top-down Rotor type SW 55 Ti Speed (g) 150000 Duration (min) 900 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 150000 Ultra filtration Cut-off size (kDa) 30 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ actin/ HSP70/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 Particle yield Not reported EM EM-type Cryo-EM Image type Wide-field EV concentration Not

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EV-TRACK ID	EV210135
species	Homo sapiens	Mus musculus
sample type	Cell culture	Cell culture
cell type	Red blood cells	B16F10
medium	Serum free medium	EV-depleted medium
condition	Control condition	Control condition
separation protocol	DG (d)(U)C UF	DG (d)(U)C UF
Exp. nr.	1	2
EV-METRIC %	78	78