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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200058	1/1	Homo sapiens	primary human macrophages derived from circulating monocytes	(d)(U)C DG	Luis A Arteaga-Blanco	2020	100%
Study summary Full title Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes All authors Luis A Arteaga-Blanco, Andrés Mojoli, Robson Q Monteiro, Vanessa Sandim, Rubem F S Menna-Barreto, Filipe Santos Pereira-Dutra, Patrícia T Bozza, Rafael de Oliveira Resende, Dumith Chequer Bou-Habib Journal PLoS One Abstract Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of t (show more...)Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes. (hide) EV-METRIC 100% (99th 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 Other / Small EVs 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 DG Protein markers EV: Alix/ CD81/ CD63/ beta actin non-EV: Calnexin/ Cytochrome c Proteomics no EV density (g/ml) 1.117- 1.181 Show all info Study aim Biomarker/protocol adaptation for the isolation and characterization of MDM-derived small EVs Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human macrophages derived from circulating monocytes EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 91 Cell count 2,00E+06 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 130 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 41 Ti Wash: speed (g) 130 000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 10% Highest density fraction 90% Total gradient volume, incl. sample (mL) 11 Sample volume (mL) 0,2 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 70 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 130 Pelleting-wash: volume per pellet (mL) 10 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) SW 41 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD63/ beta actin/ Alix/ CD81 Not detected contaminants Calnexin/ Cytochrome c Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 40-150 EV concentration Yes EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 80

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EV-TRACK ID	EV200058
species	Homo sapiens
sample type	Cell culture
cell type	primary human macrophages derived from circulating monocytes
condition	Control condition
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	100