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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190046	1/2	Homo sapiens	THP-1	(d)(U)C	Deville, Sarah	2021	56%
Study summary Full title Comparison of extracellular vesicle isolation and storage methods using high-sensitivity flow cytometry All authors Sarah Deville, Pascale Berckmans, Rebekka Van Hoof, Ivo Lambrichts, Anna Salvati, Inge Nelissen Journal PLoS One Abstract Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major (show more...)Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin LPS-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 Protein markers EV: / TSG101/ Flotillin1/ CD9 non-EV: Cytochrome C Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ TSG101 Not detected contaminants Cytochrome C Flow cytometry Type of Flow cytometry Calibration bead size Antibody details provided? No Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 182 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment A BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection was used for analysis. The device utilizes a highly sensitive fluorescence trigger to measure the EVs. Calibration bead size 0.1 EV concentration Yes

	EV190046	2/2	Homo sapiens	THP-1	exoEasy (Qiagen)	Deville, Sarah	2021	50%
Study summary Full title Comparison of extracellular vesicle isolation and storage methods using high-sensitivity flow cytometry All authors Sarah Deville, Pascale Berckmans, Rebekka Van Hoof, Ivo Lambrichts, Anna Salvati, Inge Nelissen Journal PLoS One Abstract Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major (show more...)Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day. (hide) EV-METRIC 50% (87th 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 LPS-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 exoEasy (Qiagen) Protein markers EV: / TSG101/ Flotillin1/ CD9 non-EV: Cytochrome C/ Cytochrome c Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Commercial kit Other;exoEasy (Qiagen) Other Name other separation method exoEasy (Qiagen) Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ TSG101 Not detected contaminants Cytochrome C Flow cytometry Type of Flow cytometry Calibration bead size Antibody details provided? No Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 210 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment A BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection was used for analysis. The device utilizes a highly sensitive fluorescence trigger to measure the EVs. Calibration bead size 0.1 Report type Not Reported EV concentration Yes

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EV-TRACK ID	EV190046
species	Homo sapiens
sample type	Cell culture
cell type	THP-1
condition	LPS-stimulated
separation protocol	(d)(U)C	exoEasy (Qiagen)
Exp. nr.	1	2
EV-METRIC %	56	50