Nanosep centrifugal device

For internalization assays, exosomes were isolated from BICR-18 cells (ExoB) or from AP-induced rats (ExoAP) and labelled with the PKH26 red fluorescent cell linker dye (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. The staining reaction was stopped with 3% bovine serum albumin (BSA) for 1 min. In order to remove the unbound dye, exosomes were washed three times with PBS using 300 kDa Nanosep centrifugal devices (Pall Corporation, New York, NY, USA). Fixed cells were also stained with the DNA-specific blue, fluorescent stain 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) for 1–5 min at room temperature. In some experiments, THP-1 macrophages were stained with the PKH67 green, fluorescent cell linker dye for general cell membrane.

As the presence of macromolecules gives rise to broad background signals that hinder the analysis of metabolite signals, serum specimens were filtered prior to analysis. To this end, serum samples were thawed, thoroughly shaken, and ultra-filtered employing Nanosep centrifugal devices (Pall Corporation, Port Washington, NY, USA) with a three kDa molecular weight cutoff.
Then, 400 μL of the filtrate or aqueous standard were transferred into a 5 mm NMR tube (Bruker BioSpin GmbH, Rheinstetten, Germany), followed by the addition of 200 μL of a potassium phosphate buffer at pH 7.4 and 50 μL of 0.75% (w) 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate (TSP) dissolved in deuterium oxide as an internal standard.

For tracking experiments, exosomes were labeled with PKH26 Red Fluorescent Cell Linker Dye (Sigma, St Louis, MO) according to the supplier’s specifications. The staining reaction was stopped with 3% BSA for 1 min, and the labeled exosomes (Exo-PKH26) were washed three times with PBS in order to remove the unbound dye, using 300 KDa Nanosep centrifugal devices (Pall Corporation). For each group, PBS stained with PKH26 was used as a control.
In a first tracking analysis, 7 µg of Exo-PKH26 obtained from Control or AP plasma samples were resuspended in 1 ml of saline solution and perfused through the inferior vena cava of control animals at a rate of 6 ml/h during 10 min as previously described^{9 (link)}. After 30 min, animals were sacrificed and samples of pancreas, liver, lung, kidney and small intestine were obtained and processed for the histological analysis.
In a second experiment, Exo-PKH26 from PAAF samples were perfused to control animals through the hepatic portal vein at a rate of 4 ml/h and livers from portal-perfused animals were obtained for immunofluorescence analysis.