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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV120007	1/2	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG	Lugini L	2012	78%
Study summary Full title Immune surveillance properties of human NK cell-derived exosomes All authors Lugini L, Cecchetti S, Huber V, Luciani F, Macchia G, Spadaro F, Paris L, Abalsamo L, Colone M, Molinari A, Podo F, Rivoltini L, Ramoni C, Fais S Journal J Immunol Abstract Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture s (show more...)Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by normal cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors. (hide) EV-METRIC 78% (99th 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG Adj. k-factor 41.45 (pelleting) Protein markers EV: CD63/ Rab5b/ MHC1/ CD56/ NKG2D non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.1-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation 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: time(min) 60 Pelleting: rotor type TLA100.2 Pelleting: adjusted k-factor 41.45 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Rotor type SW41 Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Rab5b/ MHC1/ CD56/ NKG2D Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Rab5b/ MHC1/ CD56/ NKG2D Characterization: Particle analysis EM EM-type immune EM Image type Close-up, Wide-field

	EV120007	2/2	Homo sapiens	Blood plasma	0.2 µm filter dUC Sucrose-DG	Lugini L	2012	67%
Study summary Full title Immune surveillance properties of human NK cell-derived exosomes All authors Lugini L, Cecchetti S, Huber V, Luciani F, Macchia G, Spadaro F, Paris L, Abalsamo L, Colone M, Molinari A, Podo F, Rivoltini L, Ramoni C, Fais S Journal J Immunol Abstract Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture s (show more...)Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by normal cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors. (hide) EV-METRIC 67% (98th 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG Adj. k-factor 37.69 (pelleting) Protein markers EV: Rab5b/ Perforin/ CD56/ NKG2D non-EV: "NKp46/ NKp44/ CD3/ CD4/ CD8" Proteomics no EV density (g/ml) 1.19-1.23 Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation 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: time(min) 120 Pelleting: rotor type TLA100.2 Pelleting: adjusted k-factor 37.69 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Rotor type SW41 Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Rab5b/ Perforin/ CD56/ NKG2D Detected contaminants "NKp46/ NKp44/ CD3/ CD4/ CD8" ELISA Detected EV-associated proteins Rab5b/ Perforin/ CD56/ NKG2D Characterization: Particle analysis

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