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You searched for: EV190095 (EV-TRACK ID)

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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 ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190095 1/2 Mus musculus Cell culture supernatant (d)(U)C
SEC
Van den Broek, Bram 2020 78%

Study summary

Full title
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% (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
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
Cell Name
BV2
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.
    • (d)(U)C = (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
Sample Condition
Control condition
EV-producing cells
BV2
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
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: 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 Cell culture supernatant (d)(U)C
SEC
Van den Broek, Bram 2020 78%

Study summary

Full title
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% (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
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
Cell Name
BV2
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.
    • (d)(U)C = (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
Sample Condition
TNFa stimulated
EV-producing cells
BV2
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
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: 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
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV190095
species
Mus musculus
sample type
Cell culture
cell type
BV2
condition
Control condition
TNFa stimulated
separation protocol
(d)(U)C
SEC
(d)(U)C
SEC
Exp. nr.
1
2
EV-METRIC %
78
78