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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220120	1/2	Homo sapiens	primary macrophages	UF qEV	Pantazi P	2022	75%
Study summary Full title Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages. All authors Pantazi P, Clements T, Venø M, Abrahams VM, Holder B Journal J Extracell Vesicles Abstract Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) ca (show more...)Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs/ we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment. (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 Cell culture supernatant Sample origin M1 pathway 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/ MHC1/ MHC2/ Fibronectin/ HSP70/ CD63/ CD81/ Syntenin-1 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary macrophages EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Ultra filtration Cut-off size (kDa) 30 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ MHC1/ MHC2/ Fibronectin Not detected EV-associated proteins HSP70 Not detected contaminants Calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ MHC1/ Syntenin-1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 162.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.25E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220120	2/2	Homo sapiens	primary macrophages	UF qEV	Pantazi P	2022	75%
Study summary Full title Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages. All authors Pantazi P, Clements T, Venø M, Abrahams VM, Holder B Journal J Extracell Vesicles Abstract Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) ca (show more...)Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs/ we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment. (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 Cell culture supernatant Sample origin M2 pathway 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/ MHC1/ MHC2/ Fibronectin/ HSP70/ CD63/ CD81/ Syntenin-1 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary macrophages EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Ultra filtration Cut-off size (kDa) 30 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ MHC1/ MHC2/ Fibronectin Not detected EV-associated proteins HSP70 Not detected contaminants Calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ MHC1/ Syntenin-1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 155.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.57E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV220120
species	Homo sapiens
sample type	Cell culture
cell type	primary macrophages
condition	M1 pathway	M2 pathway
separation protocol	Ultrafiltration/ qEV	Ultrafiltration/ qEV
Exp. nr.	1	2
EV-METRIC %	75	75