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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV160007	1/3	Homo sapiens	Blood plasma	(d)(U)C Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide) EV-METRIC 50% (81st 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 Blood plasma Sample origin Acute myeloid leukemia 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 Filtration SEC Protein markers EV: TSG101/ PD-1/ CD123/ CD96/ CD44/ CD34/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ PD-L1/ CLL-1/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 66 Western Blot Detected EV-associated proteins CD9, TSG101, CD44, CD34, CD123, CD96, CLL-1, Pro-TGFbeta1 LAP, PD-1, PD-L1 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 77-92 EV concentration Yes Particle yield 8.90E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160007	2/3	Homo sapiens	Blood plasma	(d)(U)C Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide) EV-METRIC 50% (81st 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 Blood plasma Sample origin Head and neck squamous cell carcinoma 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 Filtration SEC Protein markers EV: TSG101/ CD39/ PD-1/ CD73/ Cox2/ Fas/ FasL/ PD-L1/ HSP70/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 123 Western Blot Detected EV-associated proteins CD9, TSG101, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-1, PD-L1 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 73-76 EV concentration Yes Particle yield 1.10E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160007	3/3	Homo sapiens	Blood plasma	(d)(U)C Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide) EV-METRIC 50% (81st 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 Blood plasma 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 Filtration SEC Protein markers EV: TSG101/ CD39/ CD123/ PD-1/ CD73/ Cox2/ Fas/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ FasL/ PD-L1/ CLL-1/ HSP70/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 32 Western Blot Detected EV-associated proteins CD9, TSG101, CD123, CLL-1, Pro-TGFbeta1 LAP, PD-1, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-L1 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 69-76 EV concentration Yes Particle yield 7.00E+09 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV160007
species	Homo sapiens
sample type	Blood plasma
condition	Acute myeloid leukemia	Head and neck squamous cell carcinoma	Control condition
separation protocol	(d)(U)C Filtration SEC	(d)(U)C Filtration SEC	(d)(U)C Filtration SEC
Exp. nr.	1	2	3
EV-METRIC %	50	50	50