EV-TRACK

Search > Results

You searched for: EV230973 (EV-TRACK ID)

Showing 1 - 4 of 4

Results list Study Tree

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230973	2/4	Homo sapiens	DKs-8	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th 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 EV-dep FBS in DMEM conditioning 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 cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Albumin Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DKs-8 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.13E Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield total particles in 50 microliter: 1.14E+11

	EV230973	3/4	Homo sapiens	DLD-1	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th 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 EV-dep FBS in DMEM conditioning 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 cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Albumin Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.13E Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 157 EV concentration Yes Particle yield total particles in 50 microliter: 19470000000

	EV230973	1/4	Homo sapiens	DKs-8	(d)(U)C DC DG	Jimenez L	2023	56%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (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 (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DKs-8 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 Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.13E Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield total particles in 50 microliter: 1.24E+11

	EV230973	4/4	Homo sapiens	DLD-1	(d)(U)C DC DG	Jimenez L	2023	56%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (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 (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 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 Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.13E Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield total particles in 50 microliter: 21200000000

				1 - 4 of 4

EV-TRACK ID	EV230973
species	Homo sapiens
sample type	Cell culture
cell type	DKs-8	DLD-1	DKs-8	DLD-1
medium	EV-depleted medium	EV-depleted medium	Serum free medium	Serum free medium
condition	EV-dep FBS in DMEM conditioning	EV-dep FBS in DMEM conditioning	Control condition	Control condition
separation protocol	dUC/ DC/ Density gradient	dUC/ DC/ Density gradient	dUC/ DC/ Density gradient	dUC/ DC/ Density gradient
Exp. nr.	2	3	1	4
EV-METRIC %	67	67	56	56