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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV100021	1/2	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Lenassi M	2010	33%
Study summary Full title HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells All authors Lenassi M, Cagney G, Liao M, Vaupotic T, Bartholomeeusen K, Cheng Y, Krogan NJ, Plemenitas A, Peterlin BM Journal Traffic Abstract The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed (show more...)The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879-887). Extracellular Nef protein has deleterious effects on CD4(+) T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4(+) T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099-3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302-310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro. Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4(+) T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS). (hide) EV-METRIC 33% (70th 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 Sample origin DNF 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 Protein markers EV: Alix/ CD63/ CD81/ CD9/ TSG101/ HSC70/ LAMP2/ AChE/ Beta-actin/ Annexin2/ ICAM1/ MHC1 non-EV: Cell organelle protein Proteomics yes 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 Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD81/ CD9/ TSG101/ HSC70/ LAMP2/ AChE/ Beta-actin/ Annexin2/ ICAM1/ MHC1 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins HSC70/ LAMP2/ AChE/ Beta-actin/ Annexin2/ ICAM1/ MHC1 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV100021	2/2	Homo sapiens	Cell culture supernatant	0.2 µm filter Iodixanol-DG	Lenassi M	2010	25%
Study summary Full title HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells All authors Lenassi M, Cagney G, Liao M, Vaupotic T, Bartholomeeusen K, Cheng Y, Krogan NJ, Plemenitas A, Peterlin BM Journal Traffic Abstract The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed (show more...)The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879-887). Extracellular Nef protein has deleterious effects on CD4(+) T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4(+) T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099-3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302-310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro. Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4(+) T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS). (hide) EV-METRIC 25% (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 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 Sample origin DNF 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 + Iodixanol-DG Protein markers EV: AChE non-EV: None Proteomics no EV density (g/ml) 1.037-1.055 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 Between 800 g and 10,000 g Density gradient Density medium Iodixanol Lowest density fraction 6 Highest density fraction 18 Orientation Top-down Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins AChE ELISA Detected EV-associated proteins AChE Characterization: Particle analysis

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EV-TRACK ID	EV100021
species	Homo sapiens
sample type	Cell culture
isolation protocol	0.2 µm filter dUC	0.2 µm filter Iodixanol-DG
Exp. nr.	1	2
EV-METRIC %	33	25