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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK code 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
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% (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
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
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ CD63/ CD81/ Flotillin-1/ TSG101
Detected contaminants
Albumin/ Argonaute-2
Flow cytometry
Type of Flow cytometry
CytoFlexS
Hardware adjustments
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
Antibody details provided?
Yes
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
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% (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
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
Undetectable in void and EV fractions
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ CD63/ CD81/ Flotillin-1/ TSG101
Detected contaminants
Albumin/ Argonaute-2
Flow cytometry
Type of Flow cytometry
CytoFlexS
Hardware adjustments
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
Antibody details provided?
Yes
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
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
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