Exoquick tc ultra ev kit

Whole cell lysates were prepared using MCL1 lysis buffer in the presence of protease and phosphatase cocktail inhibitor (Sigma-Aldrich, St Louis, MO) following the manufacter's protocol. Protein lysates were subjected to SDS-PAGE on 10% polyacrylamide gel and transferred onto PVDF membranes. Membranes were probed for the protein levels of E-cadherin, and α-tubulin as loading control, using speci c primary antibodies (#4065, Cell Signaling Tech. Inc., Danvers, MA, and #sc-5286, Santa Cruz Biotechnology, Inc., Dallas, TX, respectively). Speci c bands for each protein were visualized by WesternBright™ kit (Advansta, San Jose, CA).
Enzyme-linked Immunosorbent Assay (ELISA) and human cytokine array.
Culture supernatant was collected from each growth condition, and keratinocyte growth factor (KGF-7) concentration was determined using Quantikine human KGF/FGF-7 Immunoassay (R&D Systems Inc., Minneapolis, MN). ExoQuick-TC® ULTRA EV Kit and ExoELISA-ULTRA CD63 Kit (System Biosciences, Palo Alto, CA) were used for extracellular vesicles isolation and quanti cation, respectively. For the semiquantitative detection of 23 human protein in cell culture media, the human cytokine antibody array C1 (AAH-CYT-1; RayBiotech, Norcross, GA), was used (Supplementary Figure 2). All determinations were performed according to the manufacturer's instructions.