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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100023	1/1	Mus musculus	NAY	(d)(U)C DG	Mathews JA	2010	44%
Study summary Full title CD23 Sheddase A disintegrin and metalloproteinase 10 (ADAM10) is also required for CD23 sorting into B cell-derived exosomes All authors Mathews JA, Gibb DR, Chen BH, Scherle P, Conrad DH Journal J Biol Chem Abstract The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understandin (show more...)The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understanding both the synthesis and the catabolism of CD23 are, thus, important issues. Membrane CD23 is cleaved by a disintegrin and metalloproteinase 10 (ADAM10) and this cleavage influences the ability of CD23 to regulate IgE. In contrast to the belief that cleavage is a cell surface event, endosomal neutralization with NH(4)Cl was found to dramatically reduce CD23 cleavage, suggesting that the majority of CD23 cleavage occurred subsequent to internalization in the endosomal pathway and not at the cell surface. In line with this, full-length CD23 was shown to be sorted in an ADAM10-dependent manner into exosomes. Greatly increased ADAM10-mediated CD23 cleavage was seen at endosomal pH. Additionally, the stalk region of CD23 was found to interact with ADAM10 and ADAM10 binding of CD23 was found to be protease independent. SPR analysis of the interaction indicated about a 10-fold increase in the R(max) at endosomal pH (pH 5.8) compared with pH 7.4, whereas the affinity of the interaction was not significantly changed. The R(max) change, combined with the increased cleavage at endosomal pH, indicates greater accessibility of the CD23 stalk region for ADAM10 at the lower pH. These results indicate a model where CD23 internalization results in ADAM10-dependent incorporation into exosomes, followed by partial cleavage of CD23 by ADAM10 prior to being released from the cell. The increased cleavage at endosomal pH also has implications for other ADAM10 substrates. (hide) EV-METRIC 44% (84th 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 NAY Focus vesicles exosomes 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 Adj. k-factor 124.9 (pelleting) / 124.9 (washing) Protein markers EV: ADAM10 non-EV: Proteomics no EV density (g/ml) 1.38-1.4 Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type AH650 Pelleting: adjusted k-factor 124.9 Wash: volume per pellet (ml) 5 Wash: Rotor Type AH650 Wash: adjusted k-factor 124.9 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Rotor type AH650 Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins ADAM10 ELISA Antibody details provided? No Detected EV-associated proteins ADAM10

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EV-TRACK ID	EV100023
species	Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	44