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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230381	1/1	Filifactor alocis	ATCC 35896	(d)(U)C DG Filtration UF	Kim HY	2020	57%
Study summary Full title Characterization and immunostimulatory activity of extracellular vesicles from Filifactor alocis. All authors Kim HY, Lim Y, An SJ, Choi BK Journal Mol Oral Microbiol Abstract Filifactor alocis, a gram-positive, obligate anaerobic rod, is an emerging periodontal pathogen that (show more...)Filifactor alocis, a gram-positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri-implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram-negative periodontal pathogens, so-called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP-1 and human oral keratinocyte HOK-16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270 nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter-related proteins, metabolism-related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP-1, CCL5, CXCL1, CXCL10, ICAM-1, IL-1β, IL-1ra, IL-6, IL-8, MIF, SerpinE, and TNF-α in THP-1 cells and CXCL1, G-CSF, GM-CSF, IL-6, and IL-8 in HOK-16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram-positive oral bacteria in periodontal diseases. (hide) EV-METRIC 57% (91st 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 Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Filifactor alocis Sample Type Cell culture supernatant EV-producing cells ATCC 35896 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P70AT rotor Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 10% Highest density fraction 40% Orientation Bottom-up Speed (g) 160000 Duration (min) 240 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: speed (g) 100000 Pelleting: adjusted k-factor TDB Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-270 EV concentration Yes Particle yield number of particles per million cells: 1.10E+13 EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV230381
species	Filifactor alocis
sample type	Cell culture
cell type	ATCC 35896
condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration
Exp. nr.	1
EV-METRIC %	57