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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210254	1/3	Homo sapiens	MC65	(d)(U)C Filtration	Imanbekova, Meruyert	2021	50%
Study summary Full title Identification of amyloid beta in small extracellular vesicles via Raman spectroscopy All authors Meruyert Imanbekova, Sorina Suarasan, Tatu Rojalin, Rachel R. Mizenko, Silvia Hilt, Meghna Mathur, Paula Lepine, Michael Nicouleau, Nguyen-Vi Mohamed, Thomas M. Durcan, Randy P. Carney, John C. Voss and Sebastian Wachsmann-Hogiu Journal Nanoscale advances Abstract One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and d (show more...)One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aβ) peptide into extracellular plaques. Existing research indicates that extracellular vesicles (EVs) can carry Aβ associated with AD. However, characterization of the EVs-associated Aβ and its conformational variants has yet to be realized. Raman spectroscopy is a label-free and non-destructive method that is able to assess the biochemical composition of EVs. This study reports for the first time the Raman spectroscopic fingerprint of the Aβ present in the molecular cargo of small extracellular vesicles (sEVs). Raman spectra were measured from sEVs isolated from Alzheimer's disease cell culture model, where secretion of Aβ is regulated by tetracycline promoter, and from midbrain organoids. The averaged spectra of each sEV group showed considerable variation as a reflection of the biochemical content of sEVs. Spectral analysis identified more intense Raman peaks at 1650 cm−1 and 2930 cm−1 attributable to the Aβ peptide incorporated in sEVs produced by the Alzheimer's cell culture model. Subsequent analysis of the spectra by principal component analysis differentiated the sEVs of the Alzheimer's disease cell culture model from the control groups of sEVs. Moreover, the results indicate that Aβ associated with secreted sEVs has a α-helical secondary structure and the size of a monomer or small oligomer. Furthermore, by analyzing the lipid content of sEVs we identified altered fatty acid chain lengths in sEVs that carry Aβ that may affect the fluidity of the EV membrane. Overall, our findings provide evidence supporting the use of Raman spectroscopy for the identification and characterization of sEVs associated with potential biomarkers of neurological disorders such as toxic proteins. (hide) EV-METRIC 50% (87th 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: CD9/ CD63/ CD81/ AB non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MC65 EV-harvesting Medium Serum free medium 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Wash: volume per pellet (ml) Not specified Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Filtration steps Between 0.1 and 0.2 ?m Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins AB Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 157.3 nm EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 150

	EV210254	2/3	Homo sapiens	MC65	(d)(U)C Filtration	Imanbekova, Meruyert	2021	50%
Study summary Full title Identification of amyloid beta in small extracellular vesicles via Raman spectroscopy All authors Meruyert Imanbekova, Sorina Suarasan, Tatu Rojalin, Rachel R. Mizenko, Silvia Hilt, Meghna Mathur, Paula Lepine, Michael Nicouleau, Nguyen-Vi Mohamed, Thomas M. Durcan, Randy P. Carney, John C. Voss and Sebastian Wachsmann-Hogiu Journal Nanoscale advances Abstract One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and d (show more...)One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aβ) peptide into extracellular plaques. Existing research indicates that extracellular vesicles (EVs) can carry Aβ associated with AD. However, characterization of the EVs-associated Aβ and its conformational variants has yet to be realized. Raman spectroscopy is a label-free and non-destructive method that is able to assess the biochemical composition of EVs. This study reports for the first time the Raman spectroscopic fingerprint of the Aβ present in the molecular cargo of small extracellular vesicles (sEVs). Raman spectra were measured from sEVs isolated from Alzheimer's disease cell culture model, where secretion of Aβ is regulated by tetracycline promoter, and from midbrain organoids. The averaged spectra of each sEV group showed considerable variation as a reflection of the biochemical content of sEVs. Spectral analysis identified more intense Raman peaks at 1650 cm−1 and 2930 cm−1 attributable to the Aβ peptide incorporated in sEVs produced by the Alzheimer's cell culture model. Subsequent analysis of the spectra by principal component analysis differentiated the sEVs of the Alzheimer's disease cell culture model from the control groups of sEVs. Moreover, the results indicate that Aβ associated with secreted sEVs has a α-helical secondary structure and the size of a monomer or small oligomer. Furthermore, by analyzing the lipid content of sEVs we identified altered fatty acid chain lengths in sEVs that carry Aβ that may affect the fluidity of the EV membrane. Overall, our findings provide evidence supporting the use of Raman spectroscopy for the identification and characterization of sEVs associated with potential biomarkers of neurological disorders such as toxic proteins. (hide) EV-METRIC 50% (87th 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 Tetracycline-treated 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: CD9/ CD63/ CD81/ AB non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MC65 EV-harvesting Medium Serum free medium 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Wash: volume per pellet (ml) Not specified Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Filtration steps Between 0.1 and 0.2 ?m Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins AB Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 164.1 nm EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 150

	EV210254	3/3	Homo sapiens	midbrain organoids	(d)(U)C Filtration	Imanbekova, Meruyert	2021	50%
Study summary Full title Identification of amyloid beta in small extracellular vesicles via Raman spectroscopy All authors Meruyert Imanbekova, Sorina Suarasan, Tatu Rojalin, Rachel R. Mizenko, Silvia Hilt, Meghna Mathur, Paula Lepine, Michael Nicouleau, Nguyen-Vi Mohamed, Thomas M. Durcan, Randy P. Carney, John C. Voss and Sebastian Wachsmann-Hogiu Journal Nanoscale advances Abstract One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and d (show more...)One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aβ) peptide into extracellular plaques. Existing research indicates that extracellular vesicles (EVs) can carry Aβ associated with AD. However, characterization of the EVs-associated Aβ and its conformational variants has yet to be realized. Raman spectroscopy is a label-free and non-destructive method that is able to assess the biochemical composition of EVs. This study reports for the first time the Raman spectroscopic fingerprint of the Aβ present in the molecular cargo of small extracellular vesicles (sEVs). Raman spectra were measured from sEVs isolated from Alzheimer's disease cell culture model, where secretion of Aβ is regulated by tetracycline promoter, and from midbrain organoids. The averaged spectra of each sEV group showed considerable variation as a reflection of the biochemical content of sEVs. Spectral analysis identified more intense Raman peaks at 1650 cm−1 and 2930 cm−1 attributable to the Aβ peptide incorporated in sEVs produced by the Alzheimer's cell culture model. Subsequent analysis of the spectra by principal component analysis differentiated the sEVs of the Alzheimer's disease cell culture model from the control groups of sEVs. Moreover, the results indicate that Aβ associated with secreted sEVs has a α-helical secondary structure and the size of a monomer or small oligomer. Furthermore, by analyzing the lipid content of sEVs we identified altered fatty acid chain lengths in sEVs that carry Aβ that may affect the fluidity of the EV membrane. Overall, our findings provide evidence supporting the use of Raman spectroscopy for the identification and characterization of sEVs associated with potential biomarkers of neurological disorders such as toxic proteins. (hide) EV-METRIC 50% (87th 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: CD9/ CD63/ CD81/ AB non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells midbrain organoids EV-harvesting Medium Serum free medium 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Wash: volume per pellet (ml) Not specified Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Filtration steps Between 0.1 and 0.2 ?m Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins AB Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 293.5 nm EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 150

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EV-TRACK ID	EV210254
species	Homo sapiens
sample type	Cell culture
cell type	MC65	MC65	midbrain organoids
condition	Control condition	Tetracycline-treated	Control condition
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2	3
EV-METRIC %	50	50	50