MK, AR and FN analysed the proteomics and RNA data and prepared the figures. and the transfer of their cargo to the cells can be observed. Closer inspection revealed that besides entering the EVP-6124 (Encenicline) cytoplasm, the exosomes were competent to also reach the nucleus. Furthermore, fluorescently labelled exosomal RNA enters into the cytoplasm of the MM cells. Exposure of the embryonic kidney-derived exosomes to the whole MM in an organ culture setting did not lead to an induction of nephrogenesis but had EVP-6124 (Encenicline) an impact on the overall organization of the tissue. We conclude that the exosomes provide a novel signalling system with an apparent role in secondary embryonic induction regulating organogenesis. and subsequently diluted out (to contain 1 or 10% FBS) and filtered through a 0.2?m filter (Whatman). Residual EV contamination was not found, since no EV markers were found when applied to a Western blot as a control. Following the collection of the CM, cell cultures were trypsinized, the cells were counted, and cell viability was measured on an Automatic Cell Counter (BioRad) using a 0.1% trypan blue exclusion test. The CM from pUB cells was harvested after 24C48?h of cell culture. Subsequently it was concentrated by filtration (Amicon Ultra, Millipore, 100K filters) from ~5?mL to 350?L, and stored at ?20C until usage. OptiPrep? density gradient centrifugation C exosome purification A discontinuous iodixanol gradient was used as described earlier  with some modifications. OptiPrep? density gradient (Sigma) was formed Rabbit polyclonal to RAD17 by layering 2.5?mL of 40%, 2.5?mL of 20%, 2.5?mL of 10% and 2.2?mL of 5% solutions on top of each other in a 12?mL open top polyallomer tube (Thermo Fisher). Five hundred microlitres of CM sample was overlaid onto the top of the gradient, which was then centrifuged for 18?h at 100?000?and 4C (SW 32.1 Ti rotor, Beckman Coulter). Gradient fractions of 1 1?mL were collected and tested for vesicle markers on an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently on Western blot. The fractions that contained vesicles (up to three fractions) were pooled, diluted to 45?mL in PBS and centrifuged for 3?h at 100?000?and 4C. The resulting pellets were resuspended in 1?mL of PBS and stored at ?20C. The density of each fraction was estimated according to a standard curve measuring the absorbance values at 340?nm of 1 1:100 aqueous dilutions of 5, 10, 20 and 40% iodixanol solutions. The obtained standard curve was used to determine the density of fractions collected from a control gradient overlaid with 500?L of PBS, and for the calculation of the density of each vesicle-containing fraction. Protein analysis Quantification and Western blot To estimate the amount of proteins in EX samples, a bicinchoninic acid assay (BCA assay; Pierce? BCA Protein Assay Kit) was performed according to the manufacturers recommendations. Absorbance was measured at 562?nm. Protein samples for SDS-PAGE were run at the following concentrations: for exosomes samples and all cell lysates, 5?g, for the CM from pUB 20?L was applied. The following primary and secondary antibodies were used for immunostaining: rabbit polyclonal anti-Ago2 (1:500) (#ab32381, Abcam, Cambridge, UK), mouse monoclonal anti-Alix (1:1000) (#2171, Cell Signaling, Danvers, MA), rabbit polyclonal anti-calreticulin (1:1000) (#2891, Cell Signaling), mouse monoclonal anti-CD81 (B-11) (1:400) (#sc-166029, Santa Cruz Biotechnology, Dallas, TX), rabbit EVP-6124 (Encenicline) polyclonal anti-Hsc70 (1:2000) (#ab137808, EVP-6124 (Encenicline) Abcam), mouse monoclonal anti-CD63 (LAMP-3, clone R5G2) (1:2000) (MBL, Nagoya, Japan) and mouse monoclonal anti-TG101 (1:1000) (#sc-7964, Santa Cruz Biotechnology). Secondary antibodies coupled to horseradish peroxidase were obtained from Dako (Glostrup, Denmark). Proteomics and data analysis Protein data were analysed using Proteome.