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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV170046	1/2	Sus scrofa	Cell culture supernatant	DG (d)(U)C UF	Klingeborn M	2017	88%
Study summary Full title Directional Exosome Proteomes Reflect Polarity-Specific Functions in Retinal Pigmented Epithelium Monolayers. All authors Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C Journal Sci Rep Abstract The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide) EV-METRIC 88% (98th 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 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 Cell Name Primary retinal pigmented epithelium cells Sample origin Basolateral side of polarized layer Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C UF Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: TSG101/ Syntenin-1 non-EV: Calreticulin Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Cell culture supernatant Sample Condition Basolateral side of polarized layer EV-producing cells Primary retinal pigmented epithelium cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Wash: adjusted k-factor 253.9 Density gradient Only used for validation of main results Yes Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 1 Orientation Bottom-up (sample migrates upwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Pierce 660nm Protein Assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101, Syntenin-1 Not detected contaminants Calreticulin Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 132.2±13.1 EM EM-type Transmission-EM Image type Wide-field

	EV170046	2/2	Sus scrofa	Cell culture supernatant	DG (d)(U)C UF	Klingeborn M	2017	88%
Study summary Full title Directional Exosome Proteomes Reflect Polarity-Specific Functions in Retinal Pigmented Epithelium Monolayers. All authors Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C Journal Sci Rep Abstract The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide) EV-METRIC 88% (98th 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 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 Cell Name Primary retinal pigmented epithelium cells Sample origin Apical side of polarized layer Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C UF Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: TSG101/ Syntenin-1 non-EV: Calreticulin Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Cell culture supernatant Sample Condition Apical side of polarized layer EV-producing cells Primary retinal pigmented epithelium cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Wash: adjusted k-factor 253.9 Density gradient Only used for validation of main results Yes Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 1 Orientation Bottom-up (sample migrates upwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Pierce 660nm Protein Assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101, Syntenin-1 Not detected contaminants Calreticulin Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 118.6±9.9 EM EM-type Transmission-EM Image type Close-up

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