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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170012	1/3	Homo sapiens	adipose tissue mesenchymal stromal cells	(d)(U)C	Gualerzi, Alice	2017	56%
Study summary Full title Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells All authors Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica Journal Scientific Reports Abstract Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (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 Adj. k-factor 158.5 (pelleting) / 158.5 (washing) Protein markers EV: Flotillin-1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adipose tissue mesenchymal stromal 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 55.2 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 158.5 Wash: time (min) 70 Wash: Rotor Type Type 55.2 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 158.5 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9, CD63, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 46.5 ± 15.8 Other particle analysis name(1) Raman spectroscopy Extra information Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA

	EV170012	2/3	Homo sapiens	dermal fibroblasts	(d)(U)C	Gualerzi, Alice	2017	56%
Study summary Full title Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells All authors Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica Journal Scientific Reports Abstract Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (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 Adj. k-factor 158.5 (pelleting) / 158.5 (washing) Protein markers EV: Flotillin-1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells dermal fibroblasts 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 55.2 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 158.5 Wash: time (min) 70 Wash: Rotor Type Type 55.2 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 158.5 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9, CD63, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 46.5 ± 15.8 Other particle analysis name(1) Raman spectroscopy Extra information Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA

	EV170012	3/3	Homo sapiens	bone marrow-derived mesenchymal stromal cells	(d)(U)C	Gualerzi, Alice	2017	56%
Study summary Full title Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells All authors Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica Journal Scientific Reports Abstract Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (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 Adj. k-factor 158.5 (pelleting) / 158.5 (washing) Protein markers EV: Flotillin-1/ CD63/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bone marrow-derived mesenchymal stromal 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 55.2 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 158.5 Wash: time (min) 70 Wash: Rotor Type Type 55.2 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 158.5 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9, CD63, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 46.5 ± 15.8 Other particle analysis name(1) Raman spectroscopy Extra information Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA

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EV-TRACK ID	EV170012
species	Homo sapiens
sample type	Cell culture
cell type	adipose tissue mesenchymal stromal cells	dermal fibroblasts	bone marrow-derived mesenchymal stromal cells
condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	1	2	3
EV-METRIC %	56	56	56