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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130033	1/2	Homo sapiens	Ascites	(d)(U)C DG	Bretz NP	2013	22%
Study summary Full title Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-like receptor signaling All authors Bretz NP, Ridinger J, Rupp AK, Rimbach K, Keller S, Rupp C, Marmé F, Umansky L, Umansky V, Eigenbrod T, Sammar M, Altevogt P Journal J Biol Chem Abstract Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the unde (show more...)Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1?, TNF-?, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NF?B and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NF?B and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases. (hide) EV-METRIC 22% (59th 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 Ascites Sample origin NAY Focus vesicles exosomes 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 DG Protein markers EV: Annexin1/ HSP70/ TSG101/ LAMP1 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Ascites 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) 120 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101/ Annexin1/ LAMP1 ELISA Antibody details provided? No Detected EV-associated proteins Annexin1/ LAMP1 Characterization: Particle analysis NTA

	EV130033	2/2	Homo sapiens	Amniotic fluid	(d)(U)C DG	Bretz NP	2013	22%
Study summary Full title Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-like receptor signaling All authors Bretz NP, Ridinger J, Rupp AK, Rimbach K, Keller S, Rupp C, Marmé F, Umansky L, Umansky V, Eigenbrod T, Sammar M, Altevogt P Journal J Biol Chem Abstract Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the unde (show more...)Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1?, TNF-?, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NF?B and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NF?B and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases. (hide) EV-METRIC 22% (25th 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 Amniotic fluid Sample origin NAY Focus vesicles exosomes 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 DG Protein markers EV: Annexin1/ HSP70/ TSG101/ LAMP1 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Amniotic fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101/ Annexin1/ LAMP1 ELISA Antibody details provided? No Detected EV-associated proteins Annexin1/ LAMP1 Characterization: Particle analysis NTA

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EV-TRACK ID	EV130033
species	Homo sapiens
sample type	Ascites	Amniotic fluid
condition	NAY	NAY
separation protocol	(d)(U)C DG	(d)(U)C DG
Exp. nr.	1	2
EV-METRIC %	22	22