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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100073	1/1	Homo sapiens	NAY	(d)(U)C	Bu HF	2010	14%
Study summary Full title Toll-like receptor 2-mediated peptidoglycan uptake by immature intestinal epithelial cells from apical side and exosome-associated transcellular transcytosis All authors Bu HF, Wang X, Tang Y, Koti V, Tan XD Journal J Cell Physiol Abstract Peptidoglycan (PGN) is a potent immune adjuvant derived from bacterial cell walls. Previous investig (show more...)Peptidoglycan (PGN) is a potent immune adjuvant derived from bacterial cell walls. Previous investigations suggest that intestinal epithelium may absorb PGN from the lumen. Nonetheless, how PGN is taken up and crosses intestinal epithelium remains largely unclear. Here, we first characterized PGN transport in vitro using IEC-18 and HT29-CL19A cells, which represent less mature epithelial cells in intestinal crypts. With fluorescent microscopy, we visualized internalization of dual-labeled PGN by enterocytes. Engulfed PGN was found to form a complex with PGN recognition protein-3, which may facilitate delivering PGN in vivo. Utilizing electronic microscopy, we revealed that uptake of apical PGN across intestinal epithelial monolayers was involved in phagocytosis, multivesicular body formation, and exosome secretion. We also studied transport of PGN using the transwell system. Our data indicated that apically loaded PGN was exocytosed to the basolateral compartment with exosomes by HT29-CL19A cells. The PGN-contained basolateral exosome extracts induced macrophage activation. Through gavaging mice with labeled PGN, we found that luminal PGN was taken up by columnar epithelial cells in crypts of the small intestine. Furthermore, we showed that pre-confluent immature but not post-confluent mature C2BBe1 cells engulfed PGN via a toll-like receptor 2-dependent manner. Together, our findings suggest that (1) crypt-based immature intestinal epithelial cells play an important role in transport of luminal PGN over the intestinal epithelium; and (2) luminal PGN is transcytosed across intestinal epithelia via a toll-like receptor 2-mediated phagocytosis-multivesicular body-exosome pathway. The absorbed PGN and its derivatives may facilitate maintenance of intestinal immune homeostasis. (hide) EV-METRIC 14% (44th 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 Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens 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 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) 60 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: Rotor Type 70Ti Wash: adjusted k-factor 156.9

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EV-TRACK ID	EV100073
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	14