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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170032	1/2	Mus musculus	DC2.4, 4T1	(d)(U)C Flow cytometry	Aizea Morales-Kastresana	2019	66%
Study summary Full title High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer All authors Aizea Morales-Kastresana, Thomas A. Musich, Joshua A. Welsh, William Telford, Thorsten Demberg, James C. S. Wood, Marty Bigos, Carley D. Ross, Aliaksander Kachynski, Alan Dean, Edward J. Felton, Jonathan Van Dyke, John Tigges, Vasilis Toxavidis, David R. Parks, W. Roy Overton, Aparna H. Kesarwala, Gordon J. Freeman, Ariel Rosner, Stephen P. Perfetto, Lise Pasquet, Masaki Terabe, Katherine McKinnon, Veena Kapoor, Jane B. Trepel, Anu Puri, Hisataka Kobayashi, Bryant Yung, Xiaoyuan Chen, Peter Guion, Peter Choyke, Susan J. Knox, Ionita Ghiran, Marjorie Robert-Guroff, Jay A. Berzofsky and Jennifer C. Jones Journal J Extracell Vesicles Abstract Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to man (show more...)Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the “reference noise”). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity. (hide) EV-METRIC 66% (93rd 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 Flow cytometry Adj. k-factor 156.9 (pelleting) / 41.45 (washing) Protein markers EV: Alix/ TSG101/ MHC2/ PSMA non-EV: None Proteomics no Show all info Study aim Function, New methodological development, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells DC2.4, 4T1 EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability (%) NA 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) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type TLA-120.1 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Fluorescence-activated vesicle sorting Type of flow cytometer Astrios-EQ, using combinations of protein, membrane, and epitope-specific labels Hardware adaptation to ~100nm EV's Yes Size of calibration beads (µm) 0.1, 0.2 Fluorescent labeling Specific labelling of EV conte Other Name other separation method Flow cytometry EV-subtype Used subtypes Yes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix, TSG101 Flow cytometry specific beads Selected surface protein(s) PSMA Flow cytometry Type of Flow cytometry Astrios-EQ Hardware adaptation to ~100nm EV's configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1, 0.2, 0.5 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-180 EV concentration Yes Particle yield 5.00E+11 particles/ml start sample Particle analysis: flow cytometry Flow cytometer type Astrios-EQ Hardware adjustment configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1;0.2;0.5 and various Report type Median EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information EVs purified with the serial ultracentrifugation methods were labeled with small molecules or antibodies, and excess/unbound small molecules and antibodies were removed with either NAP-5 or qEV size exclusion chromatography

	EV170032	2/2	Homo sapiens	PC3	(d)(U)C Flow cytometry	Aizea Morales-Kastresana	2019	66%
Study summary Full title High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer All authors Aizea Morales-Kastresana, Thomas A. Musich, Joshua A. Welsh, William Telford, Thorsten Demberg, James C. S. Wood, Marty Bigos, Carley D. Ross, Aliaksander Kachynski, Alan Dean, Edward J. Felton, Jonathan Van Dyke, John Tigges, Vasilis Toxavidis, David R. Parks, W. Roy Overton, Aparna H. Kesarwala, Gordon J. Freeman, Ariel Rosner, Stephen P. Perfetto, Lise Pasquet, Masaki Terabe, Katherine McKinnon, Veena Kapoor, Jane B. Trepel, Anu Puri, Hisataka Kobayashi, Bryant Yung, Xiaoyuan Chen, Peter Guion, Peter Choyke, Susan J. Knox, Ionita Ghiran, Marjorie Robert-Guroff, Jay A. Berzofsky and Jennifer C. Jones Journal J Extracell Vesicles Abstract Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to man (show more...)Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the “reference noise”). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity. (hide) EV-METRIC 66% (93rd 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 Flow cytometry Adj. k-factor 156.9 (pelleting) / 41.45 (washing) Protein markers EV: Alix/ TSG101/ MHC2/ PSMA non-EV: None Proteomics no Show all info Study aim Function, New methodological development, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability (%) NA 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) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type TLA-120.1 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Fluorescence-activated vesicle sorting Type of flow cytometer Astrios-EQ, using combinations of protein, membrane, and epitope-specific labels Hardware adaptation to ~100nm EV's Yes Size of calibration beads (µm) 0.1, 0.2 Fluorescent labeling Specific labelling of EV conte Other Name other separation method Flow cytometry EV-subtype Used subtypes Yes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix, TSG101 Flow cytometry specific beads Selected surface protein(s) PSMA Flow cytometry Type of Flow cytometry Astrios-EQ Hardware adaptation to ~100nm EV's configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1, 0.2, 0.5 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-180 EV concentration Yes Particle yield 5.00E+11 particles/ml start sample Particle analysis: flow cytometry Flow cytometer type Astrios-EQ Hardware adjustment configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1;0.2;0.5 and various Report type Median EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information EVs purified with the serial ultracentrifugation methods were labeled with small molecules or antibodies, and excess/unbound small molecules and antibodies were removed with either NAP-5 or qEV size exclusion chromatography

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EV-TRACK ID	EV170032
species	Mus musculus	Homo sapiens
sample type	Cell culture	Cell culture
cell type	DC2.4 4T1	PC3
condition	Control condition	Control condition
separation protocol	(d)(U)C Flow cytometry	(d)(U)C Flow cytometry
Exp. nr.	1	2
EV-METRIC %	66	66