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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210262	1/2	Homo sapiens	hiPSC-derived RPE	(d)(U)C DG Filtration	Flores-Bellver, Miguel	2021	89%
Study summary Full title Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors All authors Miguel Flores-Bellver, Jason Mighty, Silvia Aparicio-Domingo, Kang V Li, Cui Shi, Jing Zhou, Hannah Cobb, Patrick McGrath, German Michelis, Patricia Lenhart, Ganna Bilousova, Søren Heissel, Michael J Rudy, Christina Coughlan 10 , Andrew E Goodspeed 11 12 , S Patricia Becerra, Stephen Redenti 13 , M Valeria Canto-Soler Journal J Extracell Vesicles Abstract Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key con (show more...)Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth. (hide) EV-METRIC 89% (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. 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 (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 7.66 (washing) Protein markers EV: CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 non-EV: GM130 Proteomics yes EV density (g/ml) 1.064 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC-derived RPE EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 99 Cell count 8400000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 10 Wash: time (min) 90 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 120000 Wash: adjusted k-factor 7.66E Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type SW 41 Ti Speed (g) 141000 Duration (min) 3600 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 7.66E Filtration steps 0.2 or 0.22 ?m Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 15 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 Detected contaminants GM130 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00e+10 EM EM-type Transmission-EM Image type Close-up Report size (nm) 120

	EV210262	2/2	Homo sapiens	hiPSC-derived RPE	(d)(U)C DG Filtration	Flores-Bellver, Miguel	2021	89%
Study summary Full title Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors All authors Miguel Flores-Bellver, Jason Mighty, Silvia Aparicio-Domingo, Kang V Li, Cui Shi, Jing Zhou, Hannah Cobb, Patrick McGrath, German Michelis, Patricia Lenhart, Ganna Bilousova, Søren Heissel, Michael J Rudy, Christina Coughlan 10 , Andrew E Goodspeed 11 12 , S Patricia Becerra, Stephen Redenti 13 , M Valeria Canto-Soler Journal J Extracell Vesicles Abstract Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key con (show more...)Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth. (hide) EV-METRIC 89% (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. 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 Cigarette smoke treatment 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 (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 7.66 (washing) Protein markers EV: CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 non-EV: GM130 Proteomics yes EV density (g/ml) 1.064 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC-derived RPE EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 99 Cell count 8400000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 10 Wash: time (min) 90 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 120000 Wash: adjusted k-factor 7.66E Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type SW 41 Ti Speed (g) 141000 Duration (min) 3600 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 7.66E Filtration steps 0.2 or 0.22 ?m Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 15 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 Detected contaminants GM130 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.70e+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 120

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EV-TRACK ID	EV210262
species	Homo sapiens
sample type	Cell culture
cell type	hiPSC-derived RPE
condition	Control condition	Cigarette smoke treatment
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration
Exp. nr.	1	2
EV-METRIC %	89	89