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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210179	2/6	Mus musculus	Renca	(d)(U)C DG	Samoylenko, Anatoliy	2021	88%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (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 (d)(U)C DG Protein markers EV: TSG101/ Alix/ CD9/ CD81 non-EV: Argonaute2/ GM130 Proteomics no EV density (g/ml) 1.07-1.12 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 2 Orientation Top-down Rotor type TH-641 Speed (g) 100000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 0.17 Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101/ Alix/ CD81 Not detected contaminants GM130/ Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes Particle yield Yes, as number of particles per million cells 9.96E+06 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV210179	4/6	Mus musculus	Renca	(d)(U)C DG	Samoylenko, Anatoliy	2021	88%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (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 hypoxia 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 DG Protein markers EV: Alix/ TSG101/ CD9/ CD81 non-EV: Argonaute2/ GM130 Proteomics no EV density (g/ml) 1.07-1.12 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 2 Orientation Top-down Rotor type TH-641 Speed (g) 100000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 0.185 Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ TSG101/ CD81 Not detected contaminants GM130/ Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 128 EV concentration Yes Particle yield Yes, as number of particles per million cells 2.10E+07 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV210179	1/6	Mus musculus	Renca	(d)(U)C Exo-Spin	Samoylenko, Anatoliy	2021	67%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (hide) EV-METRIC 67% (94th 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 Commercial method Protein markers EV: CD81/ TSG101/ CD9/ Alix non-EV: Argonaute2/ GM130 Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Commercial kit Exo-Spin Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per million cells 1.15 Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101/ Alix/ CD81 Not detected contaminants GM130/ Argonaute2 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131 EV concentration Yes Particle yield Yes, as number of particles per million cells 9.07E+07 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV210179	3/6	Mus musculus	Renca	(d)(U)C Exo-Spin	Samoylenko, Anatoliy	2021	67%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (hide) EV-METRIC 67% (94th 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 hypoxia 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 Commercial method Protein markers EV: CD81/ TSG101/ CD9/ Alix non-EV: Argonaute2/ GM130 Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Commercial kit Exo-Spin Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 1.53 Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ TSG101/ CD81 Not detected contaminants GM130/ Argonaute2 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168 EV concentration Yes Particle yield Yes, as number of particles per million cells 1.73E+08 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV210179	5/6	Homo sapiens	786-O	(d)(U)C Exo-Spin	Samoylenko, Anatoliy	2021	56%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (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 (d)(U)C Commercial method Protein markers EV: CD81 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 786-O EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Commercial kit Exo-Spin Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 1.18 Western Blot Antibody details provided? No Detected EV-associated proteins CD81 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 177.5 EV concentration Yes Particle yield Yes, as number of particles per million cells 3.74E+07 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-200

	EV210179	6/6	Homo sapiens	786-O	(d)(U)C Exo-Spin	Samoylenko, Anatoliy	2021	56%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (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 hypoxia 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 Commercial method Protein markers EV: CD81 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 786-O EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Commercial kit Exo-Spin Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 1.43 Western Blot Antibody details provided? No Detected EV-associated proteins CD81 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 153.5 EV concentration Yes Particle yield Yes, as number of particles per million cells 7.27E+07 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-200

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EV-TRACK ID	EV210179
species	Mus musculus	Mus musculus	Mus musculus	Mus musculus	Homo sapiens	Homo sapiens
sample type	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture
cell type	Renca	Renca	Renca	Renca	786-O	786-O
condition	Control condition	hypoxia	Control condition	hypoxia	Control condition	hypoxia
separation protocol	(d)(U)C DG	(d)(U)C DG	(d)(U)C Exo-Spin	(d)(U)C Exo-Spin	(d)(U)C Exo-Spin	(d)(U)C Exo-Spin
Exp. nr.	2	4	1	3	5	6
EV-METRIC %	88	88	67	67	56	56