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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200058	1/1	Homo sapiens	primary human macrophages derived from circulating monocytes	(d)(U)C DG	Luis A Arteaga-Blanco	2020	100%
Study summary Full title Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes All authors Luis A Arteaga-Blanco, Andrés Mojoli, Robson Q Monteiro, Vanessa Sandim, Rubem F S Menna-Barreto, Filipe Santos Pereira-Dutra, Patrícia T Bozza, Rafael de Oliveira Resende, Dumith Chequer Bou-Habib Journal PLoS One Abstract Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of t (show more...)Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / Small EVs 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 (d)(U)C DG Protein markers EV: Alix/ CD81/ CD63/ beta actin non-EV: Calnexin/ Cytochrome c Proteomics no EV density (g/ml) 1.117- 1.181 Show all info Study aim Biomarker/protocol adaptation for the isolation and characterization of MDM-derived small EVs Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human macrophages derived from circulating monocytes EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 91 Cell count 2,00E+06 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 130 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 41 Ti Wash: speed (g) 130 000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 10% Highest density fraction 90% Total gradient volume, incl. sample (mL) 11 Sample volume (mL) 0,2 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 70 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 130 Pelleting-wash: volume per pellet (mL) 10 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) SW 41 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD63/ beta actin/ Alix/ CD81 Not detected contaminants Calnexin/ Cytochrome c Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 40-150 EV concentration Yes EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 80

	EV190105	1/2	Homo sapiens	Human umbilical vein endothelial cells (HUVECs)	DG (d)(U)C	Emanuela Mensà	2020	100%
Study summary Full title Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells All authors Emanuela Mensà, Michele Guescini, Angelica Giuliani, Maria Giulia Bacalini, Deborah Ramini, Giacomo Corleone, Manuela Ferracin, Gianluca Fulgenzi, Laura Graciotti, Francesco Prattichizzo, Leonardo Sorci, Michela Battistelli, Vladia Monsurrò, Anna Rita Bonfigli, Maurizio Cardelli, Rina Recchioni, Fiorella Marcheselli, Silvia Latini, Serena Maggio, Mirco Fanelli, Stefano Amatori, Gianluca Storci, Antonio Ceriello, Vilberto Stocchi, Maria De Luca, Luca Magnani, Maria Rita Rippo, Antonio Domenico Procopio, Claudia Sala, Iva Budimir, Cristian Bassi, Massimo Negrini, Paolo Garagnani, Claudio Franceschi, Jacopo Sabbatinelli, Massimiliano Bonafè, Fabiola Olivieri Journal J Extracell Vesicles Abstract The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. H (show more...)The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: TSG101/ Calnexin/ CD63 non-EV: Albumin Proteomics no EV density (g/ml) 1.08-1.10 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Human umbilical vein endothelial cells (HUVECs) EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 103745 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type Type 90 Ti Wash: speed (g) 109354 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type SW 28 Speed (g) 103745 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3.5 Pelleting: duration (min) 70 Pelleting: rotor type Type 90 Ti Pelleting: speed (g) 109354 Pelleting-wash: volume per pellet (mL) 3 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) Type 90 Ti Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ TSG101/ Calnexin Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 20 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 80 (sEVs) / 120 (lEVs) EV concentration Yes Particle yield Number of particles per cell: 3E4 (sEVs)/1.5E4 (lEVs) EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190103	1/1	Homo sapiens	HaCaT	DG (d)(U)C Dynabeads CD9, CD63, CD81	Xiaoju Zhou	2020	100%
Study summary Full title Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing All authors Xiaoju Zhou, Brooke A Brown, Amanda P Siegel, Mohamed El Masry, Xuyao Zeng, Woran Song, Amitava Das, Puneet Khandelwal, Andrew Clark, Kanhaiya Singh, Poornachander R Guda, Mahadeo Gorain, Lava Timsina, Yi Xuan, Stephen C Jacobson, Milos V Novotny, Sashwati Roy, Mangilal Agarwal, Robert J Lee, Chandan K Sen, David E Clemmer, Subhadip Ghatak Journal ACS Nano Abstract Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a (show more...)Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP reporter (Exoκ-GFP) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by wound-macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFP in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, TLNPκ/si-hnRNPA2B1 treated group showed significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP are recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties post-injury. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Dynabeads CD9, CD63, CD81 Protein markers EV: TSG101/ Alix non-EV: Proteomics no EV density (g/ml) 1.16 Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HaCaT EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 95 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) 120 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 6 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 1.05 Sample volume (mL) 0.05 Orientation Top-down Rotor type TLA-120.2 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.1 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 120 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Commercial kit Dynabeads CD9, CD63, CD81 Other Name other separation method Dynabeads CD9, CD63, CD81 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins TSG101/ Alix/ FLOT1/ HSP90 Detected contaminants Prohibitin Not detected contaminants GM130 Flow cytometry specific beads Detected EV-associated proteins TSG101 Other 1 Fluorescent anisotropy Detected EV-associated proteins TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 102 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.20E+07 EM EM-type Scanning-EM Image type Wide-field

	EV190097	1/2	Homo sapiens	Synovial fluid	(d)(U)C SEC	Foers, Andrew	2020	100%
Study summary Full title Proteomic analysis of extracellular vesicles reveals an immunogenic cargo in rheumatoid arthritis synovial fluid All authors Andrew D Foers, Laura F Dagley, Simon Chatfield, Andrew I Webb, Lesley Cheng, Andrew F Hill, Ian P Wicks, Ken C Pang Journal Clinical & Translational Immunology Abstract Results: Synovial fluid EVs were present at higher concentrations in RA joints with high‐level inf (show more...)Results: Synovial fluid EVs were present at higher concentrations in RA joints with high‐level inflammation (P‐value = 0.004) but were smaller in diameter (P‐value = 0.03) than in low‐level inflammation. In total, 1058 SF EV proteins were identified by mass spectrometry analysis. Neutrophil and fibroblast markers were overrepresented in all disease groups. Numerous proteins with potential to modulate inflammatory and immunological processes were detected, including nine citrullinated peptides. Forty‐five and 135 EV‐associated proteins were significantly elevated in RA joints with high‐level inflammation than in RA joints with low‐level inflammation and OA joints, respectively. Gene ontology analysis revealed significant enrichment for proteins associated with ‘neutrophil degranulation’ within SF EVs from RA joints with high‐level inflammation. Conclusion: Our results provide new information about SF EVs and insight into how EVs might contribute to the perpetuation of RA. (hide) EV-METRIC 100% (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 Synovial fluid Sample origin Inflamed rheumatoid arthritis joints 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 (d)(U)C SEC Protein markers EV: non-EV: Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Synovial fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-45 Pelleting: speed (g) 58100 Size-exclusion chromatography Total column volume (mL) 320 Sample volume/column (mL) 5 Resin type Sephacryl S-500 HR Characterization: Protein analysis PMID previous EV protein analysis PMID: 29963299 Protein Concentration Method BCA Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190097	2/2	Homo sapiens	Synovial fluid	(d)(U)C SEC	Foers, Andrew	2020	100%
Study summary Full title Proteomic analysis of extracellular vesicles reveals an immunogenic cargo in rheumatoid arthritis synovial fluid All authors Andrew D Foers, Laura F Dagley, Simon Chatfield, Andrew I Webb, Lesley Cheng, Andrew F Hill, Ian P Wicks, Ken C Pang Journal Clinical & Translational Immunology Abstract Results: Synovial fluid EVs were present at higher concentrations in RA joints with high‐level inf (show more...)Results: Synovial fluid EVs were present at higher concentrations in RA joints with high‐level inflammation (P‐value = 0.004) but were smaller in diameter (P‐value = 0.03) than in low‐level inflammation. In total, 1058 SF EV proteins were identified by mass spectrometry analysis. Neutrophil and fibroblast markers were overrepresented in all disease groups. Numerous proteins with potential to modulate inflammatory and immunological processes were detected, including nine citrullinated peptides. Forty‐five and 135 EV‐associated proteins were significantly elevated in RA joints with high‐level inflammation than in RA joints with low‐level inflammation and OA joints, respectively. Gene ontology analysis revealed significant enrichment for proteins associated with ‘neutrophil degranulation’ within SF EVs from RA joints with high‐level inflammation. Conclusion: Our results provide new information about SF EVs and insight into how EVs might contribute to the perpetuation of RA. (hide) EV-METRIC 100% (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 Synovial fluid Sample origin Non-inflamed rheumatoid arthritis joints 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 (d)(U)C SEC Protein markers EV: non-EV: Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Synovial fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-45 Pelleting: speed (g) 58100 Size-exclusion chromatography Total column volume (mL) 320 Sample volume/column (mL) 5 Resin type Sephacryl S-500 HR Characterization: Protein analysis PMID previous EV protein analysis PMID: 29963299 Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 210 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190064	5/10	Homo sapiens	Urine	DG (d)(U)C UF	Dhondt B	2020	100%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine 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 DG (d)(U)C UF Protein markers EV: TSG101/ Alix/ Flotillin1/ CD9 non-EV: Tamm-Horsfall protein Proteomics no EV density (g/ml) 1.087-1.109 Show all info Study aim Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD9/ TSG101/ Alix Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 196.5 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV190064	6/10	Homo sapiens	Urine	DG (d)(U)C UF	Dhondt B	2020	100%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine 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 DG (d)(U)C UF Protein markers EV: TSG101/ Alix/ Flotillin1/ CD9 non-EV: Tamm-Horsfall protein Proteomics no EV density (g/ml) 1.087-1.109 Show all info Study aim Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD9/ TSG101/ Alix Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131.7 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV190064	7/10	Homo sapiens	Urine	DG (d)(U)C UF	Dhondt B	2020	100%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Prostate Cancer 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 DG (d)(U)C UF Protein markers EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1 non-EV: Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.087-1.109 Show all info Study aim Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9 Detected contaminants Tamm-Horsfall protein Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV190064	8/10	Homo sapiens	Urine	DG (d)(U)C UF	Dhondt B	2020	100%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Bladder Cancer 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 DG (d)(U)C UF Protein markers EV: Alix/ Flotillin1/ CD9/ Syntenin-1 non-EV: Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.087-1.109 Show all info Study aim Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ Syntenin-1/ CD9 Detected contaminants Tamm-Horsfall protein Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV190064	9/10	Homo sapiens	Urine	DG (d)(U)C UF	Dhondt B	2020	100%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Renal Cancer 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 DG (d)(U)C UF Protein markers EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1 non-EV: Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.087-1.109 Show all info Study aim Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9 Detected contaminants Tamm-Horsfall protein Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-300 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV190064	10/10	Homo sapiens	Tissue	DG (d)(U)C UF	Dhondt B	2020	100%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 100% (92nd 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 Tissue Sample origin Prostate Cancer 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 DG (d)(U)C UF Protein markers EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1 non-EV: Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.087-1.109 Show all info Study aim Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9 Detected contaminants Tamm-Horsfall protein Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150.3 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV190044	11/11	Homo sapiens	Blood plasma	DG (d)(U)C qEV	Driedonks, Tom A.P.	2020	100%
Study summary Full title Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases All authors Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen Journal J Extracell Vesicles Abstract Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 DG (d)(U)C qEV Protein markers EV: CD81/ CD63/ Flotillin1/ CD9 non-EV: ApoAl/ ApoB100 Proteomics no EV density (g/ml) 1.11 - 1.18 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.4 M Highest density fraction 2.5 M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.05 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 192000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ CD81 Not detected contaminants ApoAl/ ApoB100 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 0.045 RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.16 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 130 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190006	1/2	Homo sapiens	MDAMB231	DG (d)(U)C	Altei, Wanessa F.	2020	100%
Study summary Full title Inhibition of αvβ3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles All authors Wanessa F Altei, Bianca C Pachane, Patty K Dos Santos, Lígia N M Ribeiro, Bong Hwan Sung, Alissa M Weaver, Heloisa S Selistre-de-Araújo Journal Cell Commun Signal Abstract Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from (show more...)Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion: In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin immortalized 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 Density gradient (Differential) (ultra)centrifugation Protein markers EV: Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1 non-EV: Calnexin Proteomics no EV density (g/ml) 1.11 Show all info Study aim Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Cell viability (%) 95 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 4 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 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) 3 Orientation Bottom-up Rotor type Type 40 Speed (g) 100000 Duration (min) 18 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 180 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 3 Pelleting-wash: duration (min) 180 Pelleting-wash: speed (g) TLA-100.3 Density cushion Density medium EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1 Not detected EV-associated proteins Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 107 EV concentration Yes Particle yield Yes, as number of particles per million cells 5.50E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm)

	EV200031	4/8	Homo sapiens	Blood plasma	DG (d)(U)C	Grossi, Ilaria	2020	89%
Study summary Full title MicroRNA‑34a‑5p expression in the plasma and in its extracellular vesicle fractions in subjects with Parkinson's disease: An exploratory study All authors Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro Journal Int J Mol Med Abstract Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4 non-EV: Apo-AI/ GM130 Proteomics no EV density (g/ml) 1.09-1.22 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 15% Highest density fraction 70% Total gradient volume, incl. sample (mL) 4.4 Sample volume (mL) 1 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.4 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81 Not detected contaminants Apo-AI/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase H RNAse concentration 0.00625 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force microscopy Image type Close-up, Wide-field Report size (nm) 50-100 Report type Not Reported

	EV200031	8/8	Homo sapiens	Blood plasma	DG (d)(U)C	Grossi, Ilaria	2020	89%
Study summary Full title MicroRNA‑34a‑5p expression in the plasma and in its extracellular vesicle fractions in subjects with Parkinson's disease: An exploratory study All authors Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro Journal Int J Mol Med Abstract Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Parkinson's disease 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 DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4 non-EV: Apo-AI/ GM130 Proteomics no EV density (g/ml) 1.09-1.22 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 15% Highest density fraction 70% Total gradient volume, incl. sample (mL) 4.4 Sample volume (mL) 1 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.4 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81 Not detected contaminants Apo-AI/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase H RNAse concentration 0.00625 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force microscopy Image type Close-up, Wide-field Report size (nm) 50-100 Report type Not Reported

	EV200015	1/4	Homo sapiens	primary human dermal fibroblasts	DG (d)(U)C Filtration	Streck, Nicholas	2020	89%
Study summary Full title Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread All authors Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich Journal J Virol Abstract Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Protein markers EV: TSG101/ CD81/ CD63 non-EV: Tubulin Proteomics no EV density (g/ml) Density not calculated Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human dermal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 130000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 5% Highest density fraction 41% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 130000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 130000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected contaminants Tubulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 170 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.01E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report type Not Reported EV-concentration No

	EV200015	2/4	Homo sapiens	primary human dermal fibroblasts	DG (d)(U)C Filtration	Streck, Nicholas	2020	89%
Study summary Full title Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread All authors Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich Journal J Virol Abstract Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin HCMV infected 72hpi 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD81/ HCMV glycoprotein B/ CD63 non-EV: Tubulin Proteomics no EV density (g/ml) Density not calculated Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human dermal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 130000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 5% Highest density fraction 41% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 130000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 130000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ HCMV glycoprotein B/ TSG101/ CD81 Not detected contaminants Tubulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 158 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.51E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report type Not Reported EV-concentration No

	EV200001	1/9	Homo sapiens	EBC1	DG (d)(U)C	Useckaite, Zivile	2020	89%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells EBC1 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 99 Cell count 2.25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Density gradient Type Continuous Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 1080 Fraction volume (mL) 5 Fraction processing Centrifugation Pelleting: volume per fraction 17;17mL Pelleting: duration (min) 120 Pelleting: rotor type SW 32.1 Ti;SW 32 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin1/ Actinin4 Not detected contaminants GRP94 ELISA Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 111 EV concentration Yes Particle yield Yes, as number of particles per million cells 86300000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	4/9	Homo sapiens	H596	DG (d)(U)C	Useckaite, Zivile	2020	89%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Density gradient Type Continuous Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 1080 Fraction volume (mL) 5 Fraction processing Centrifugation Pelleting: volume per fraction 17;17mL Pelleting: duration (min) 120 Pelleting: rotor type SW 32.1 Ti;SW 32 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Syntenin1/ Actinin4/ CD9/ CD63 Not detected contaminants GRP94 ELISA Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 106 EV concentration Yes Particle yield Yes, as number of particles per million cells 89500000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	7/9	Homo sapiens	Hs746T	DG (d)(U)C	Useckaite, Zivile	2020	89%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Hs746T EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Density gradient Type Continuous Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 1080 Fraction volume (mL) 5 Fraction processing Centrifugation Pelleting: volume per fraction 17;17mL Pelleting: duration (min) 120 Pelleting: rotor type SW 32.1 Ti;SW 32 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Syntenin1/ Actinin4/ CD9/ CD63 Not detected contaminants GRP94 ELISA Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 107 EV concentration Yes Particle yield Yes, as number of particles per million cells 29000000000 EM EM-type Transmission-EM Image type Close-up

	EV200073	2/4	Homo sapiens	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC DG	Bordas, Marie	2020	88%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 88% (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 Solid Tissue Sample origin CLL 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 (d)(U)C Size-exclusion chromatopraphy (IZON) DC DG Protein markers EV: TSG101/ CD81/ Flotillin1/ CD9 non-EV: cytochrome C/ GM130/ Calreticulin Proteomics no EV density (g/ml) 1.12 Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 0% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 7 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100 Duration (min) 120 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ CD9/ TSG101/ CD81 Detected contaminants Calreticulin Not detected contaminants cytochrome C/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.00E+10 EM EM-type Immuno-EM/ Transmission-EM EM protein Other;HLA-DR Image type Close-up

	EV200073	4/4	Mus musculus	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC DG	Bordas, Marie	2020	88%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 88% (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 Solid Tissue Sample origin CLL 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 (d)(U)C Size-exclusion chromatopraphy (IZON) DC DG Protein markers EV: Alix/ TSG101/ Flotillin1 non-EV: ATPA5/ Calreticulin Proteomics no EV density (g/ml) 1.12 Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 0% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 7 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100 Duration (min) 120 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ Alix/ TSG101 Detected contaminants Calreticulin Not detected contaminants ATPA5 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 7.00E+09 EM EM-type Transmission-EM Image type Close-up

	EV180012	1/1	Mus musculus	4T1	DG (d)(U)C Filtration UF	Van Deun, Jan	2020	87%
Study summary Full title Feasibility of Mechanical Extrusion to Coat Nanoparticles with Extracellular Vesicle Membranes All authors Jan Van Deun, Quentin Roux, Sarah Deville, Thibaut Van Acker, Pekka Rappu, Ilkka Miinalainen, Jyrki Heino, Frank Vanhaecke, Bruno G De Geest, Olivier De Wever, An Hendrix Journal Cells Abstract Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field (show more...)Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field of intense study. Extracellular vesicles (EV), sub-micron delivery vehicles for intercellular communication, have unique characteristics for drug delivery. We investigated the top-down functionalization of gold nanoparticles with extracellular vesicle membranes, including both lipids and associated membrane proteins, through mechanical extrusion. EV surface-exposed membrane proteins were confirmed to help avoid unwanted elimination by macrophages, while improving autologous uptake. EV membrane morphology, protein composition and orientation were found to be unaffected by mechanical extrusion. We implemented complementary EV characterization methods, including transmission- and immune-electron microscopy, and nanoparticle tracking analysis, to verify membrane coating, size and zeta potential of the EV membrane-cloaked nanoparticles. While successful EV membrane coating of the gold nanoparticles resulted in lower macrophage uptake, low yield was found to be a significant downside of the extrusion approach. Our data incentivize more research to leverage EV membrane biomimicking as a unique drug delivery approach in the near future. (hide) EV-METRIC 87% (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. 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 DG (d)(U)C Filtration UF Protein markers EV: Alix/ TSG101/ Flotillin1/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis PMID previous EV protein analysis Proteomics Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix, TSG101, CD9, Flotillin1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 113.4±2.8 EV concentration Yes EM EM-type Transmission-EM/ Immune-EM EM protein CD9 Image type Close-up, Wide-field

	EV230049	1/2	Homo sapiens	Brain gray matter	(d)(U)C DG Filtration	Muraoka S	2020	78%
Study summary Full title Proteomic and biological profiling of extracellular vesicles from Alzheimer's disease human brain tissues. All authors Muraoka S, DeLeo AM, Sethi MK, Yukawa-Takamatsu K, Yang Z, Ko J, Hogan JD, Ruan Z, You Y, Wang Y, Medalla M, Ikezu S, Chen M, Xia W, Gorantla S, Gendelman HE, Issadore D, Zaia J, Ikezu T Journal Alzheimers Dement Abstract Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta ( (show more...)Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aβ) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined. (hide) EV-METRIC 78% (50th 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 Brain gray matter Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: AB1-40/ AB1-42/ ANXA-5 non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain gray matter 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.475 M Highest density fraction 2.0 M Total gradient volume, incl. sample (mL) 14 Sample volume (mL) 2 Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 1200 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA ELISA Detected EV-associated proteins AB1-40/ AB1-42/ ANXA-5 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 131 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV230049	2/2	Homo sapiens	Brain gray matter	(d)(U)C DG Filtration	Muraoka S	2020	78%
Study summary Full title Proteomic and biological profiling of extracellular vesicles from Alzheimer's disease human brain tissues. All authors Muraoka S, DeLeo AM, Sethi MK, Yukawa-Takamatsu K, Yang Z, Ko J, Hogan JD, Ruan Z, You Y, Wang Y, Medalla M, Ikezu S, Chen M, Xia W, Gorantla S, Gendelman HE, Issadore D, Zaia J, Ikezu T Journal Alzheimers Dement Abstract Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta ( (show more...)Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aβ) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined. (hide) EV-METRIC 78% (50th 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 Brain gray matter Sample origin Alzheimer's disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: AB1-40/ AB1-42/ ANXA5 non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain gray matter 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.475 M Highest density fraction 2.0 M Total gradient volume, incl. sample (mL) 14 Sample volume (mL) 2 Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 1200 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA ELISA Detected EV-associated proteins AB1-40/ AB1-42/ ANXA5 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 122 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV200065	1/4	Homo sapiens	DLD1	(d)(U)C Filtration	Victoria Stary	2020	78%
Study summary Full title Short-course radiotherapy promotes pro-inflammatory macrophages via extracellular vesicles in human rectal cancer All authors Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md Journal Methods & Clinical Development Abstract Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (hide) EV-METRIC 78% (97th 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 (d)(U)C Filtration Protein markers EV: CD9/ CD81/ TSG101 non-EV: Calreticulin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >=100,000g Cell viability (%) 85 Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type T-1250 Pelleting: speed (g) 243836 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type SW 41 Ti Wash: speed (g) 92,500 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 132 EV concentration Yes EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200065	2/4	Homo sapiens	DLD1	(d)(U)C Filtration	Victoria Stary	2020	78%
Study summary Full title Short-course radiotherapy promotes pro-inflammatory macrophages via extracellular vesicles in human rectal cancer All authors Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md Journal Methods & Clinical Development Abstract Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (hide) EV-METRIC 78% (97th 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 gamma irradiation 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 (d)(U)C Filtration Protein markers EV: CD9/ CD81/ TSG101 non-EV: Calreticulin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >=100,000g Cell viability (%) 85 Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type T-1250 Pelleting: speed (g) 243836 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type SW 41 Ti Wash: speed (g) 92,500 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 137 EV concentration Yes EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200065	3/4	Homo sapiens	HCT116	(d)(U)C Filtration	Victoria Stary	2020	78%
Study summary Full title Short-course radiotherapy promotes pro-inflammatory macrophages via extracellular vesicles in human rectal cancer All authors Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md Journal Methods & Clinical Development Abstract Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (hide) EV-METRIC 78% (97th 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 (d)(U)C Filtration Protein markers EV: CD9/ CD81/ TSG101 non-EV: Calreticulin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >=100,000g Cell viability (%) 85 Cell count 4.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type T-1250 Pelleting: speed (g) 243836 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type SW 41 Ti Wash: speed (g) 92,500 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 157 EV concentration Yes EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200065	4/4	Homo sapiens	HCT116	(d)(U)C Filtration	Victoria Stary	2020	78%
Study summary Full title Short-course radiotherapy promotes pro-inflammatory macrophages via extracellular vesicles in human rectal cancer All authors Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md Journal Methods & Clinical Development Abstract Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (hide) EV-METRIC 78% (97th 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 gamma irradiation 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 (d)(U)C Filtration Protein markers EV: CD9/ CD81/ TSG101 non-EV: Calreticulin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >=100,000g Cell viability (%) 85 Cell count 4.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type T-1250 Pelleting: speed (g) 243836 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type SW 41 Ti Wash: speed (g) 92,500 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 161 EV concentration Yes EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200036	1/16	Homo sapiens	human skin primary fibroblasts	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	78%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 78% (97th 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 Young donors 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 DG (d)(U)C Commercial method Protein markers EV: Alix/ TSG101/ GSTM2 non-EV: Calnexin/ Actin-beta Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human skin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ TSG101/ GSTM2 Not detected contaminants Calnexin/ Actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV200036	3/16	Homo sapiens	human skin primary fibroblasts	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	78%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 78% (97th 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 Old donors 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 DG (d)(U)C Commercial method Protein markers EV: Alix/ TSG101/ GSTM2 non-EV: Calnexin/ Actin-beta Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human skin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ TSG101/ GSTM2 Not detected contaminants Calnexin/ Actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV190095	1/2	Mus musculus	BV2	(d)(U)C SEC	Van den Broek, Bram	2020	78%
Study summary Full title Microglial derived extracellular vesicles activate autophagy and mediate multi‐target signaling to maintain cellular homeostasis All authors Bram Van den Broek, Isabel Pintelon, Ibrahim Hamad, Sofie Kessels, Mansour Haidar, Niels Hellings, Jerome J.A. Hendriks, Markus Kleinewietfeld, Bert Brône, Vincent Timmerman, Jean‐Pierre Timmermans, Veerle Somers, Luc Michiels, Joy Irobi Journal J Extracell Vesicles Abstract Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in (show more...)Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial‐derived extracellular vesicles (M‐EVs) to the maintenance of microglia homeostasis and how M‐EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M‐EVs is limited. Here, we analysed the effects of M‐EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non‐activated microglia BV2 cells. We showed that M‐EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M‐EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M‐EVs increased the protein expression of the autophagy marker, microtubule‐associated proteins 1A/1B light chain 3B isoform II (LC3B‐II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M‐EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M‐EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia‐microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis. (hide) EV-METRIC 78% (97th 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 (d)(U)C SEC Protein markers EV: CD81/ Flotillin1/ Annexin A2 non-EV: GRP94 Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells BV2 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 115000 Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ Annexin A2/ CD81 Not detected contaminants GRP94 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV190095	2/2	Mus musculus	BV2	(d)(U)C SEC	Van den Broek, Bram	2020	78%
Study summary Full title Microglial derived extracellular vesicles activate autophagy and mediate multi‐target signaling to maintain cellular homeostasis All authors Bram Van den Broek, Isabel Pintelon, Ibrahim Hamad, Sofie Kessels, Mansour Haidar, Niels Hellings, Jerome J.A. Hendriks, Markus Kleinewietfeld, Bert Brône, Vincent Timmerman, Jean‐Pierre Timmermans, Veerle Somers, Luc Michiels, Joy Irobi Journal J Extracell Vesicles Abstract Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in (show more...)Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial‐derived extracellular vesicles (M‐EVs) to the maintenance of microglia homeostasis and how M‐EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M‐EVs is limited. Here, we analysed the effects of M‐EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non‐activated microglia BV2 cells. We showed that M‐EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M‐EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M‐EVs increased the protein expression of the autophagy marker, microtubule‐associated proteins 1A/1B light chain 3B isoform II (LC3B‐II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M‐EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M‐EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia‐microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis. (hide) EV-METRIC 78% (97th 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 TNFa stimulated 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 (d)(U)C SEC Protein markers EV: CD81/ Flotillin1/ Annexin A2 non-EV: GRP94 Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells BV2 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 115000 Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ Annexin A2/ CD81 Not detected contaminants GRP94 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV190084	1/2	Homo sapiens	Dental pulp stromal cells	(d)(U)C Filtration	Greet Merckx	2020	78%
Study summary Full title Angiogenic Effects of Human Dental Pulp and Bone Marrow-Derived Mesenchymal Stromal Cells and Their Extracellular Vesicles All authors Greet Merckx, Baharak Hosseinkhani, Sören Kuypers, Sarah Deville, Joy Irobi, Inge Nelissen, Luc Michiels, Ivo Lambrichts, Annelies Bronckaers Journal Cells Abstract Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marr (show more...)Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis. (hide) EV-METRIC 78% (97th 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 (d)(U)C Filtration Protein markers EV: CD9/ CD63/ ANXA2/ CD81 non-EV: Bax Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Dental pulp stromal cells EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ ANXA2/ CD81 Not detected contaminants Bax

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