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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210339	1/2	Homo sapiens	Blood plasma	UF qEV Original 70nm	Sandau US	2020	75%
Study summary Full title Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study. All authors Sandau US, Duggan E, Shi X, Smith SJ, Huckans M, Schutzer WE, Loftis JM, Janowsky A, Nolan JP, Saugstad JA Journal J Extracell Vesicles Abstract Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neur (show more...)Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neurotoxicity and cardiovascular disease. Recent studies have begun to link microRNAs (miRNAs) to the processes related to MA use and addiction. Our studies are the first to analyse plasma EVs and their miRNA cargo in humans actively using MA (MA-ACT) and control participants (CTL). In this cohort we also assessed the effects of tobacco use on plasma EVs. We used vesicle flow cytometry to show that the MA-ACT group had an increased abundance of EV tetraspanin markers (CD9, CD63, CD81), but not pro-coagulant, platelet-, and red blood cell-derived EVs. We also found that of the 169 plasma EV miRNAs, eight were of interest in MA-ACT based on multiple statistical criteria. In smokers, we identified 15 miRNAs of interest, two that overlapped with the eight MA-ACT miRNAs. Three of the MA-ACT miRNAs significantly correlated with clinical features of MA use and target prediction with these miRNAs identified pathways implicated in MA use, including cardiovascular disease and neuroinflammation. Together our findings indicate that MA use regulates EVs and their miRNA cargo, and support that further studies are warranted to investigate their mechanistic role in addiction, recovery, and recidivism. (hide) EV-METRIC 75% (96th 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 Ultrafiltration Commercial method Protein markers EV: Alix/ CD9/ CD63/ CD81/ Flotillin?1/ TSG101 non-EV: Albumin/ Argonaute-2 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit qEV Original 70nm Other Name other separation method Commercial method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ CD81/ Flotillin-1/ TSG101 Detected contaminants Albumin/ Argonaute-2 Flow cytometry Type of Flow cytometry CytoFlexS Hardware adaptation to ~100nm EV's The CytoFlex flow cytometer with stock filters was configured to measure violet side scatter (VSSC) as described in the CytoFLEX Instructions for Use (https://www.beckman.com/techdocs/B49006AP/wsr-168786). Briefly, the Violet 405nm filter is placed in position 2, the Violet 450nm filter in position 3, and an unused filter in position 1. The gain on all scatter channels was set to 100, the gain on all fluorescence channels was set to 1000. Calibration bead size vCal nanoRainbow, Cellarcus (500nm)/ Quantum FITC Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 40-200nm Particle analysis: flow cytometry Flow cytometer type CytoFlexS Hardware adjustment The CytoFlex flow cytometer with stock filters was configured to measure violet side scatter (VSSC) as described in the CytoFLEX Instructions for Use (https://www.beckman.com/techdocs/B49006AP/wsr-168786). Briefly, the Violet 405nm filter is placed in position 2, the Violet 450nm filter in position 3, and an unused filter in position 1. The gain on all scatter channels was set to 100, the gain on all fluorescence channels was set to 1000. Calibration bead size vCal nanoRainbow, Cellarcus (500nm)/ Quantum FITC, Bangs Labs/ Quantibrite PE, BD Biosciences Report type Mean Reported size (nm) 75-400nm EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00e+10 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 50-200nm

	EV210339	2/2	Homo sapiens	Blood plasma	UF qEV Original 70nm	Sandau US	2020	75%
Study summary Full title Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study. All authors Sandau US, Duggan E, Shi X, Smith SJ, Huckans M, Schutzer WE, Loftis JM, Janowsky A, Nolan JP, Saugstad JA Journal J Extracell Vesicles Abstract Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neur (show more...)Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neurotoxicity and cardiovascular disease. Recent studies have begun to link microRNAs (miRNAs) to the processes related to MA use and addiction. Our studies are the first to analyse plasma EVs and their miRNA cargo in humans actively using MA (MA-ACT) and control participants (CTL). In this cohort we also assessed the effects of tobacco use on plasma EVs. We used vesicle flow cytometry to show that the MA-ACT group had an increased abundance of EV tetraspanin markers (CD9, CD63, CD81), but not pro-coagulant, platelet-, and red blood cell-derived EVs. We also found that of the 169 plasma EV miRNAs, eight were of interest in MA-ACT based on multiple statistical criteria. In smokers, we identified 15 miRNAs of interest, two that overlapped with the eight MA-ACT miRNAs. Three of the MA-ACT miRNAs significantly correlated with clinical features of MA use and target prediction with these miRNAs identified pathways implicated in MA use, including cardiovascular disease and neuroinflammation. Together our findings indicate that MA use regulates EVs and their miRNA cargo, and support that further studies are warranted to investigate their mechanistic role in addiction, recovery, and recidivism. (hide) EV-METRIC 75% (96th 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 Active methamphetamine use disorder 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 Ultrafiltration Commercial method Protein markers EV: Alix/ CD9/ CD63/ CD81/ Flotillin?1/ TSG101 non-EV: Albumin/ Argonaute-2 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit qEV Original 70nm Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Undetectable in void and EV fractions Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ CD81/ Flotillin-1/ TSG101 Detected contaminants Albumin/ Argonaute-2 Flow cytometry Type of Flow cytometry CytoFlexS Hardware adaptation to ~100nm EV's The CytoFlex flow cytometer with stock filters was configured to measure violet side scatter (VSSC) as described in the CytoFLEX Instructions for Use (https://www.beckman.com/techdocs/B49006AP/wsr-168786). Briefly, the Violet 405nm filter is placed in position 2, the Violet 450nm filter in position 3, and an unused filter in position 1. The gain on all scatter channels was set to 100, the gain on all fluorescence channels was set to 1000. Calibration bead size vCal nanoRainbow, Cellarcus (500nm)/ Quantum FITC Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 40-200nm Particle analysis: flow cytometry Flow cytometer type CytoFlexS Hardware adjustment The CytoFlex flow cytometer with stock filters was configured to measure violet side scatter (VSSC) as described in the CytoFLEX Instructions for Use (https://www.beckman.com/techdocs/B49006AP/wsr-168786). Briefly, the Violet 405nm filter is placed in position 2, the Violet 450nm filter in position 3, and an unused filter in position 1. The gain on all scatter channels was set to 100, the gain on all fluorescence channels was set to 1000. Calibration bead size vCal nanoRainbow, Cellarcus (500nm)/ Quantum FITC, Bangs Labs/ Quantibrite PE, BD Biosciences Report type Mean Reported size (nm) 75-400nm EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00e+10 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 50-200nm

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EV-TRACK ID	EV210339
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	Active methamphetamine use disorder
separation protocol	Ultrafiltration/ qEV Original 70nm	Ultrafiltration/ qEV Original 70nm
Exp. nr.	1	2
EV-METRIC %	75	75