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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180030	3/7	Homo sapiens	PM1	DG (d)(U)C	Zhaohao Liao	2018	67%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 67% (94th 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 HIV-BaL infected 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 DG (d)(U)C Protein markers EV: / CD81/ CD63/ CD9 non-EV: AChE Proteomics no EV density (g/ml) Not specified Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PM1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type TH-641 Speed (g) 210000 Duration (min) 120 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 40 Pelleting: rotor type AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Other 1 Acetylcholinesterase assay Detected EV-associated proteins Not detected contaminants AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV180030	5/7	Homo sapiens	Jurkat	(d)(U)C qEV	Zhaohao Liao	2018	56%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 56% (90th 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 qEV Protein markers EV: / CD63 non-EV: AChE Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants AChE Other 1 Acetylcholinesterase assay Detected EV-associated proteins Detected contaminants AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein Other;AChE Image type Close-up, Wide-field

	EV180030	6/7	Homo sapiens	Jurkat	DG (d)(U)C	Zhaohao Liao	2018	56%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 56% (90th 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 pseudotyped HIV-1 (NL4-3) infected 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 DG (d)(U)C Protein markers EV: / CD63 non-EV: p24/ AChE Proteomics no EV density (g/ml) Not specified Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 60 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 40 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants p24 Not detected contaminants AChE Other 1 Acetylcholinesterase assay Detected EV-associated proteins Not detected contaminants AChE Characterization: Lipid analysis No

	EV180030	2/7	Homo sapiens	H9	(d)(U)C qEV	Zhaohao Liao	2018	33%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 33% (74th 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 qEV Protein markers EV: / CD81/ CD63 non-EV: AChE Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H9 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD81 Other 1 Acetylcholinesterase assay Detected EV-associated proteins Detected contaminants AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV180030	1/7	Homo sapiens	U937	(d)(U)C	Zhaohao Liao	2018	29%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 29% (67th 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 Protein markers EV: non-EV: AChE Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U937 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Protein Concentration Method Not determined Other 1 Acetylcholinesterase assay Detected EV-associated proteins Detected contaminants AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV180030	4/7	Homo sapiens	PM1	(d)(U)C	Zhaohao Liao	2018	29%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 29% (67th 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 Protein markers EV: non-EV: AChE Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PM1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Protein Concentration Method Not determined Other 1 Acetycholinesterase assay Detected EV-associated proteins Detected contaminants AChE Characterization: Lipid analysis No

	EV180030	7/7	Homo sapiens	primary red blood cells	(d)(U)C qEV	Zhaohao Liao	2018	22%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 22% (58th 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 qEV Protein markers EV: AChE/ CD63 non-EV: Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary red blood cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Other 1 Acetylcholinesterase assay Detected EV-associated proteins AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

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EV-TRACK ID	EV180030
species	Homo sapiens
sample type	Cell culture
cell type	PM1	Jurkat	Jurkat	H9	U937	PM1	primary red blood cells
condition	HIV-BaL infected	Control condition	pseudotyped HIV-1 (NL4-3) infected	Control condition	Control condition	Control condition	Control condition
separation protocol	DG (d)(U)C	(d)(U)C qEV	DG (d)(U)C	(d)(U)C qEV	(d)(U)C	(d)(U)C	(d)(U)C qEV
Exp. nr.	3	5	6	2	1	4	7
EV-METRIC %	67	56	56	33	29	29	22

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