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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230062	5/6	Mus musculus	Blood plasma	(d)(U)C Filtration	Choi YY	2023	63%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 63% (93rd 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 Blood plasma 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 Filtration Protein markers EV: CD9/ CD63/ CD81/ CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA -sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 85 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~35000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230062	1/6	Homo sapiens	HUVEC/THP1 (coculture)	(d)(U)C Filtration UF	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (86th 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 Filtration Ultrafiltration Protein markers EV: CD31/ CD105/ CD144/ CD146 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC/THP1 (coculture) EV-harvesting Medium Serum-containing medium Cell viability (%) 90 Cell count 4000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Flow cytometry Type of Flow cytometry conventional flow cytometer Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 119 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~2.5E06 EM EM-type Transmission-EM Image type Wide-field

	EV230062	2/6	Homo sapiens	HUVEC/THP1 (coculture)	(d)(U)C Filtration UF	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (86th 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 Irradiation of co-culture (1 Gy) 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 Filtration Ultrafiltration Protein markers EV: CD31/ CD105/ CD144/ CD146 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC/THP1 (coculture) EV-harvesting Medium Serum-containing medium Cell viability (%) 90 Cell count 4000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 112 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~5E06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230062	3/6	Homo sapiens	Blood plasma	(d)(U)C Filtration	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (85th 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 Blood plasma 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 Filtration Protein markers EV: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 82 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~32000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230062	4/6	Homo sapiens	Blood plasma	(d)(U)C Filtration	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (85th 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 Blood plasma Sample origin Irradiation of blood sample (1 Gy) 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 Filtration Protein markers EV: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 88 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~34000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230062	6/6	Mus musculus	Blood plasma	(d)(U)C Filtration	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (85th 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 Blood plasma Sample origin Irradiation of whole body (1 Gy) 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 Filtration Protein markers EV: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA -sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 71 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~39000 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV230062
species	Mus musculus	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Mus musculus
sample type	Blood plasma	Cell culture	Cell culture	Blood plasma	Blood plasma	Blood plasma
cell type	NA	HUVEC/THP1 (coculture)	HUVEC/THP1 (coculture)	NA	NA	NA
medium	NA	Serum-containing medium	Serum-containing medium	NA	NA	NA
condition	Control condition	Control condition	Irradiation of co-culture (1 Gy)	Control condition	Irradiation of blood sample (1 Gy)	Irradiation of whole body (1 Gy)
separation protocol	dUC/ Filtration	dUC/ Filtration/ Ultrafiltration	dUC/ Filtration/ Ultrafiltration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
Exp. nr.	5	1	2	3	4	6
EV-METRIC %	63	50	50	50	50	50