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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	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

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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