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Results list Most used isolation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV160001	1/1	Mus musculus	Cell culture supernatant	DG dUC	Stremersch S	2016	87%
Study summary Full title Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs. All authors Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC, Raemdonck K Journal J Control Release Abstract Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natu (show more...)Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier. (hide) EV-METRIC 87% (99th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosome-like vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Protein markers EV: CD63/ CD81/ HSP70/ beta-actin non-EV: Calreticulin/ GM130 Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16F10, JAWSII EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS Ultrafiltration (MWCO 300 kDa) Cell viability (%) 95 Isolation Method Differential ultra centrifugation Differential UC: filtering steps Below or equal to 800 g Between 10,000 g and 50,000 g Density gradient Only used for validation of main results Yes Density medium 115.5 Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 0.125 Highest density fraction 0.5 Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) Rotor type SW 55 Ti Speed (g) 200000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 115.5 Pelleting-wash: volume per pellet (mL) 5 Pelleting-wash: duration (min) 70 Pelleting-wash: rotor type 115.5 Pelleting-wash: speed (g) SW 55 Ti Pelleting-wash: adjusted k-factor 115.5 EV-subtype Used subtypes NO Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, HSP70, beta-actin Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-300 EV concentration Yes Particle yield 1000 EM EM-type Cryo-EM Image type Close-up

	EV160004	2/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	66%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 66% (80th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Synovial fluid Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 71.08 (pelleting) Protein markers EV: Annexin-A1/ CD90/Thy1.1 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Origin Control condition Isolation Method Differential ultra centrifugation Differential UC: filtering steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 120 Pelleting: rotor type MLS-50 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 71.08 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0M Highest density fraction 1.4M Sample volume (mL) 1.5 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 60 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 138.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Annexin-A1, CD90/Thy1.1 Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 20-200 Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.

	EV160004	5/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	66%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 66% (80th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Synovial fluid Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 1421 (pelleting) Protein markers EV: Annexin-A1/ CD90/Thy1.1 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Origin Control condition Isolation Method Differential ultra centrifugation Differential UC: filtering steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 120 Pelleting: rotor type MLS-50 Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 1421. Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0M Highest density fraction 1.4M Sample volume (mL) 1.5 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 60 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 138.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Annexin-A1, CD90/Thy1.1 Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 20-200 Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.

	EV160005	1/2	Homo sapiens	Bronchoalveolar lavage fluid	dUC Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (50th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Bronchoalveolar lavage fluid Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Origin Control condition Isolation Method Differential ultra centrifugation Differential UC: filtering steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 138.1±2.2 EV concentration Yes Particle yield 2.48E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

	EV160005	2/2	Homo sapiens	Bronchoalveolar lavage fluid	dUC Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (50th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Bronchoalveolar lavage fluid Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Origin Smokers Isolation Method Differential ultra centrifugation Differential UC: filtering steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 139.8±3.3 EV concentration Yes Particle yield 1.94E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

	EV160004	1/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (30th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Synovial fluid Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 83.68 (pelleting) Protein markers EV: CD9/ CD44 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Origin Control condition Isolation Method Differential ultra centrifugation Differential UC: filtering steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Pelleting: time(min) 65 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 83.68 Density gradient Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160004	3/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (30th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Synovial fluid Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 167.3 (pelleting) Protein markers EV: CD9/ CD44 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Origin Control condition Isolation Method Differential ultra centrifugation Differential UC: filtering steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 65 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 167.3 Density gradient Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160004	4/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (30th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Synovial fluid Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 1673 (pelleting) Protein markers EV: CD9/ CD44 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Origin Control condition Isolation Method Differential ultra centrifugation Differential UC: filtering steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 35 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 1673. Density gradient Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160000	1/1	Homo sapiens	Other	DG dUC	van Herwijnen MJ	2016	50%
Study summary Full title Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components. All authors van Herwijnen MJ, Zonneveld MI, Goerdayal S, Nolte-'t Hoen EN, Garssen J, Stahl B, Maarten Altelaar AF, Redegeld FA, Wauben MH Journal Mol Cell Proteomics Abstract Breast milk contains several macromolecular components with distinctive functions, whereby milk fat (show more...)Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. (hide) EV-METRIC 50% (89th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Other Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Protein markers EV: CD9/ Flotillin-1 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Other Origin Control condition Isolation Method Differential ultra centrifugation Differential UC: filtering steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 6.5 Orientation Top-down (sample migrates downwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 38.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis PMID previous EV protein analysis 25206958 Extra characterization Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1 Proteomics database Yes Characterization: Particle analysis PMID previous EV particle analysis 25206958 Extra information A different gradient protocol was used in the publication of the same group that was referred to for additional protein and particle analysis of milk-derived extracellular vesicles (PMID: 25206958).

	EV160002	2/3	Homo sapiens	Extruded cells (NIH3T3)	DG Sequential extrusion	Lunavat TR	2016	37%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 37% (50th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Extruded cells (NIH3T3) Focus vesicles Nanovesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + Sequential extrusion Protein markers EV: PDGFR/ Flotillin-1/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (NIH3T3) Origin Control condition Isolation Method Density cushion Density medium Iodixanol Other Name other isolation method Sequential extrusion (10 µm, 5 µm, 1 µm) Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Lysis buffer provided? Yes Detected EV-associated proteins PDGFR, Flotillin-1, CD9 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-200 EM EM-type Transmission-EM Image type Wide-field

	EV160002	3/3	Homo sapiens	Extruded cells (NIH3T3)	DG Sequential extrusion	Lunavat TR	2016	37%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 37% (50th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Extruded cells (NIH3T3) Focus vesicles Nanovesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + Sequential extrusion Protein markers EV: PDGFR/ Flotillin-1/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (NIH3T3) Origin cMyc shRNA Isolation Method Density cushion Density medium Iodixanol Other Name other isolation method Sequential extrusion (10 µm, 5 µm, 1 µm) Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Lysis buffer provided? Yes Detected EV-associated proteins PDGFR, Flotillin-1, CD9 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-200 EM EM-type Transmission-EM Image type Wide-field

	EV160003	1/1	Mus musculus	Cell culture supernatant	dUC	Morales-Kastresana A	2016	14%
Study summary Full title Labeling Extracellular Vesicles for Nanoscale Flow Cytometry. All authors Morales-Kastresana A, Telford B, Musich TA, McKinnon K, Clayborne C, Braig Z, Rosner A, Demberg T, Watson DC, Karpova TS, Freeman GJ, DeKruyff RH, Pavlakis GN, Terabe M, Robert-Guroff M, Berzofsky JA, Jones JC Journal Sci Rep Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that ar (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease. (hide) EV-METRIC 14% (44th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 156.9 (pelleting) / 41.45 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells DC2.4 EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultra centrifugation Differential UC: filtering 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: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 70 Wash: Rotor Type TLA-120.1 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Protein Concentration Method BCA Protein Concentration 1.186 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-180 EV concentration Yes Particle yield 4.8E+11 particles/ml start sample Particle analysis: flow cytometry Flow cytometer type Beckman Astrios EQ Hardware adjustment NanoFACS system Calibration bead size 0.1;0.2;0.5

	EV160002	1/3	Homo sapiens	Extruded cells (U973)	DG Sequential extrusion	Lunavat TR	2016	0%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 0% (median: 0% 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Extruded cells (U973) Focus vesicles Nanovesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + Sequential extrusion Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (U973) Origin Control condition Isolation Method Density cushion Density medium Iodixanol Other Name other isolation method Sequential extrusion (10 µm, 5 µm, 1 µm) Protein Concentration Method Bradford Characterization: Particle analysis DLS Report type Modus Reported size (nm) 150

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