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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220090	1/6	Homo sapiens	wound fluid	(d)(U)C Filtration IAF UF DG	Guda PR	2023	89%
Study summary Full title Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients. All authors Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S Journal Nano Today Abstract Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (hide) EV-METRIC 89% (75th 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 wound fluid Sample origin Non-diabetic Focus vesicles exosome 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 Filtration Immunoaffinity capture (non-commercial) Ultrafiltration Density gradient Protein markers EV: HSP90/ Alix/ CD81/ Flotillin-1/ TSG101/ ANXA5/ ICAM/ CD63 non-EV: Prohibitin/ GM130 Proteomics yes EV density (g/ml) 1.15-1.2 Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type wound fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 0.8M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 0.9 Sample volume (mL) 0.15 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.15 Fraction processing Ultracentrifugation Pelleting: volume per fraction 0.5 Pelleting: duration (min) 120 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9/CD63/CD81/ KRT14 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per E08 particles: 0.55 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins HSP90 Not detected contaminants Prohibitin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? Yes Selected surface protein(s) CD9/ CD63/ CD81/ KRT14 Proteomics database No Detected EV-associated proteins Alix/ CD81/ Flotillin-1/ TSG101/ ANXA5/ ICAM Not detected EV-associated proteins CD63/ EPCAM Detected contaminants GM130 Detected EV-associated proteins CD9/C63/CD81/ KRT5 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 87 EV concentration Yes Particle yield as particles per mg of albumin: 2.30E+08 EM EM-type Scanning-EM/ Immuno-EM EM protein TSG101 Image type Close-up, Wide-field

	EV220090	2/6	Homo sapiens	wound fluid	(d)(U)C Filtration IAF UF	Guda PR	2023	14%
Study summary Full title Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients. All authors Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S Journal Nano Today Abstract Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (hide) EV-METRIC 14% (25th 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 wound fluid Sample origin Diabetic Focus vesicles exosome 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 Filtration Immunoaffinity capture (non-commercial) Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type wound fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9/CD63/CD81/ KRT14 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per E08 particles: 0.425 Proteomics database No Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 80-110 EV concentration Yes Particle yield as particles per mg of albumin: 5.00E+07

	EV220090	3/6	Homo sapiens	primary keratinocytes	(d)(U)C Filtration IAF	Guda PR	2023	14%
Study summary Full title Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients. All authors Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S Journal Nano Today Abstract Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (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. 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 exosome 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 Filtration Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary keratinocytes EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Filtration steps 0.2 or 0.22 µm Immunoaffinity capture Selected surface protein(s) CD9/CD63/CD81/ KRT14 Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 65 EV concentration Yes Particle yield as particles per mL starting sample: 8.00E+08

	EV220090	4/6	Homo sapiens	dermal fibroblasts	(d)(U)C Filtration IAF	Guda PR	2023	14%
Study summary Full title Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients. All authors Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S Journal Nano Today Abstract Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (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. 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 exosome 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 Filtration Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells dermal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Filtration steps 0.2 or 0.22 µm Immunoaffinity capture Selected surface protein(s) CD9/CD63/CD81/ KRT14 Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 64 EV concentration Yes Particle yield as particles per mL starting sample: 2.00E+08

	EV220090	5/6	Homo sapiens	dermal microvesicular endothelial cells	(d)(U)C Filtration IAF	Guda PR	2023	14%
Study summary Full title Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients. All authors Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S Journal Nano Today Abstract Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (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. 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 exosome 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 Filtration Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells dermal microvesicular endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Filtration steps 0.2 or 0.22 µm Immunoaffinity capture Selected surface protein(s) CD9/CD63/CD81/ KRT14 Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 65 EV concentration Yes Particle yield as particles per mL starting sample: 2.00E+08

	EV220090	6/6	Homo sapiens	monocyte-derived macrophages	(d)(U)C Filtration IAF	Guda PR	2023	14%
Study summary Full title Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients. All authors Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S Journal Nano Today Abstract Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (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. 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 exosome 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 Filtration Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells monocyte-derived macrophages EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Filtration steps 0.2 or 0.22 µm Immunoaffinity capture Selected surface protein(s) CD9/CD63/CD81/ KRT14 Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 112 EV concentration Yes Particle yield as particles per mL starting sample: 1.00E+08

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EV-TRACK ID	EV220090
species	Homo sapiens
sample type	wound fluid	wound fluid	Cell culture	Cell culture	Cell culture	Cell culture
cell type	NA	NA	primary keratinocytes	dermal fibroblasts	dermal microvesicular endothelial cells	monocyte-derived macrophages
medium	NA	NA	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium
condition	Non-diabetic	Diabetic	Control condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Filtration/ IAF capture (non-commercial)/ Ultrafiltration/ Density gradient	dUC/ Filtration/ IAF capture (non-commercial)/ Ultrafiltration	dUC/ Filtration/ IAF capture (non-commercial)	dUC/ Filtration/ IAF capture (non-commercial)	dUC/ Filtration/ IAF capture (non-commercial)	dUC/ Filtration/ IAF capture (non-commercial)
Exp. nr.	1	2	3	4	5	6
EV-METRIC %	89	14	14	14	14	14