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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230346	2/3	Lactobacillus plantarum	APsulloc 331261	(d)(U)C DG Filtration UF Tangential Flow Filtration	Kim W	2020	71%
Study summary Full title -derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization . All authors Kim W, Lee EJ, Bae IH, Myoung K, Kim ST, Park PJ, Lee KH, Pham AVQ, Ko J, Oh SH, Cho EG Journal J Extracell Vesicles Abstract Probiotics offer various health benefits. has been used for decades to enhance human intestinal muc (show more...)Probiotics offer various health benefits. has been used for decades to enhance human intestinal mucosal immunity and improve skin barrier integrity. Extracellular vesicles (EVs) derived from eukaryotic or prokaryotic cells have been recognized as efficient carriers for delivery of biomolecules to recipient cells, and to efficiently regulate human pathophysiology. However, the mechanism underlying the beneficial effects of probiotic bacteria-derived EVs on human skin is unclear. Herein, we investigated how -derived EVs (LEVs) exert beneficial effects on human skin by examining the effect of LEVs on cutaneous immunity, particularly on macrophage polarization. LEVs promoted differentiation of human monocytic THP1 cells towards an anti-inflammatory M2 phenotype, especially M2b, by inducing biased expression of cell-surface markers and cytokines associated with M2 macrophages. Pre- or post-treatment with LEVs under inflammatory M1 macrophage-favouring conditions, induced by LPS and interferon-γ, inhibited M1-associated surface marker, expression. Moreover, LEV treatment significantly induced expression of macrophage-characteristic cytokines, IL-1β, GM-CSF and the representative anti-inflammatory cytokine, IL-10, in human skin organ cultures. Hence, LEVs can trigger M2 macrophage polarization , and induce an anti-inflammatory phenomenon in the human skin, and may be a potent anti-inflammatory strategy to alleviate hyperinflammatory skin conditions. (hide) EV-METRIC 71% (96th 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 Density gradient Filtration Ultrafiltration Tangential Flow Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Lactobacillus plantarum Sample Type Cell culture supernatant EV-producing cells APsulloc 331261 EV-harvesting Medium Serum free medium Cell count 2.70E+09 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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: rotor type Type 45 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 20% Highest density fraction 50% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 2.5 Orientation Bottom-up Speed (g) 200000 Duration (min) 120 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type NS Other Name other separation method Tangential Flow Filtration Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 46.5 TRPS Report type Mean Reported size (nm) 83 EV concentration Yes Particle yield other:/ particles per milligram of protein: 3.83E10 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV230346	1/3	Lactobacillus plantarum	APsulloc 331261	(d)(U)C Filtration UF Tangential Flow Filtration	Kim W	2020	14%
Study summary Full title -derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization . All authors Kim W, Lee EJ, Bae IH, Myoung K, Kim ST, Park PJ, Lee KH, Pham AVQ, Ko J, Oh SH, Cho EG Journal J Extracell Vesicles Abstract Probiotics offer various health benefits. has been used for decades to enhance human intestinal muc (show more...)Probiotics offer various health benefits. has been used for decades to enhance human intestinal mucosal immunity and improve skin barrier integrity. Extracellular vesicles (EVs) derived from eukaryotic or prokaryotic cells have been recognized as efficient carriers for delivery of biomolecules to recipient cells, and to efficiently regulate human pathophysiology. However, the mechanism underlying the beneficial effects of probiotic bacteria-derived EVs on human skin is unclear. Herein, we investigated how -derived EVs (LEVs) exert beneficial effects on human skin by examining the effect of LEVs on cutaneous immunity, particularly on macrophage polarization. LEVs promoted differentiation of human monocytic THP1 cells towards an anti-inflammatory M2 phenotype, especially M2b, by inducing biased expression of cell-surface markers and cytokines associated with M2 macrophages. Pre- or post-treatment with LEVs under inflammatory M1 macrophage-favouring conditions, induced by LPS and interferon-γ, inhibited M1-associated surface marker, expression. Moreover, LEV treatment significantly induced expression of macrophage-characteristic cytokines, IL-1β, GM-CSF and the representative anti-inflammatory cytokine, IL-10, in human skin organ cultures. Hence, LEVs can trigger M2 macrophage polarization , and induce an anti-inflammatory phenomenon in the human skin, and may be a potent anti-inflammatory strategy to alleviate hyperinflammatory skin conditions. (hide) EV-METRIC 14% (43rd 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 Ultrafiltration Tangential Flow Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Lactobacillus plantarum Sample Type Cell culture supernatant EV-producing cells APsulloc 331261 EV-harvesting Medium Serum free medium Cell count 2.70E+09 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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: rotor type Type 45 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type NS Other Name other separation method Tangential Flow Filtration Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 55.5 TRPS Report type Mean Reported size (nm) 104 EV concentration Yes Particle yield other:/ particles per milligram of protein: 2.13E9

	EV230346	3/3	Staphylococcus aureus	ATCC 6538	(d)(U)C Filtration UF Tangential Flow Filtration	Kim W	2020	14%
Study summary Full title -derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization . All authors Kim W, Lee EJ, Bae IH, Myoung K, Kim ST, Park PJ, Lee KH, Pham AVQ, Ko J, Oh SH, Cho EG Journal J Extracell Vesicles Abstract Probiotics offer various health benefits. has been used for decades to enhance human intestinal muc (show more...)Probiotics offer various health benefits. has been used for decades to enhance human intestinal mucosal immunity and improve skin barrier integrity. Extracellular vesicles (EVs) derived from eukaryotic or prokaryotic cells have been recognized as efficient carriers for delivery of biomolecules to recipient cells, and to efficiently regulate human pathophysiology. However, the mechanism underlying the beneficial effects of probiotic bacteria-derived EVs on human skin is unclear. Herein, we investigated how -derived EVs (LEVs) exert beneficial effects on human skin by examining the effect of LEVs on cutaneous immunity, particularly on macrophage polarization. LEVs promoted differentiation of human monocytic THP1 cells towards an anti-inflammatory M2 phenotype, especially M2b, by inducing biased expression of cell-surface markers and cytokines associated with M2 macrophages. Pre- or post-treatment with LEVs under inflammatory M1 macrophage-favouring conditions, induced by LPS and interferon-γ, inhibited M1-associated surface marker, expression. Moreover, LEV treatment significantly induced expression of macrophage-characteristic cytokines, IL-1β, GM-CSF and the representative anti-inflammatory cytokine, IL-10, in human skin organ cultures. Hence, LEVs can trigger M2 macrophage polarization , and induce an anti-inflammatory phenomenon in the human skin, and may be a potent anti-inflammatory strategy to alleviate hyperinflammatory skin conditions. (hide) EV-METRIC 14% (43rd 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 Ultrafiltration Tangential Flow Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells ATCC 6538 EV-harvesting Medium Serum free medium Cell count 8.60E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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: rotor type Type 45 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type NS Other Name other separation method Tangential Flow Filtration Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV230346
species	Lactobacillus plantarum	Lactobacillus plantarum	Staphylococcus aureus
sample type	Cell culture	Cell culture	Cell culture
cell type	APsulloc 331261	APsulloc 331261	ATCC 6538
condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration/ Tangential Flow Filtration	dUC/ Filtration/ Ultrafiltration/ Tangential Flow Filtration	dUC/ Filtration/ Ultrafiltration/ Tangential Flow Filtration
Exp. nr.	2	1	3
EV-METRIC %	71	14	14