Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).Funding agencies: he National Cancer Institute, United States CA224175 (D.L.), CA210240 (D.L.), CA232093(D.L.), CA163117 and CA207983 (D.L.), CA163120 (D.L.), CA169416 (D.L.), CA169538 (D.L.),CA218513 (D.L., H.Z.) and AI144301 (D.L., V.P.); the United States Department of Defense, UnitedStates W81XWH-13-1-0425 (D.L.), W81XWH-13-1-0427, W81XWH-13-1-0249 (D.L.), and W81XWH-14-1-0199 (D.L.); National Institutes of Health, United States/WCM CTSC, United States (NIH/NCATS (UL1TR00457) (H.Z.); NIH/NCATS (UL1TR002384) (D.L. and H.Z.); the Hartwell Foundation,United States (D.L.); the Thompson Family Foundation (D.L., D.K.); the STARR Consortium I9-A9-056(D.L., H.Z.) and I8-A8-123 (D.L.); the Pediatric Oncology Experimental Therapeutics Investigator’sConsortium (D.L., T.M.T.); Alex’s Lemonade Stand Foundation, United Sates (D.L.); the Breast Can-cer Research Foundation, United States (D.L.); the Feldstein Medical Foundation (D.L.); the TortolaniFoundation (D.L.); the Clinical &amp; Translational Science Center (D.L., H.Z.); the Mary Kay Ash Chari-table Foundation (D.L., I.M.); the Malcolm Hewitt Weiner Foundation (D.L.); the Manning Founda-tion (DL., A.H.); the Daniel P. and Nancy C. Paduano Family Foundation (D.L); the James PaduanoFoundation (D.L.); the Sohn Foundation (D.L.); the AHEPA Vth District Cancer Research Foundation(D.L., L.B., S.L.); the Daedalus Fund Selma and Lawrence Ruben Science to Industry Bridge Award(D.L.); the Children’s Cancer and Blood Foundation, United States (D.L.); Susan G. Komen Postdoc-toral Fellowship PDF15331556, JST PRESTO, Japan 30021, and JSPS KAKENHI, Japan JP19K23743(A.H.); the National Research Foundation of Korea, South Korea (NRF) grant funded by the Korea government (MSIT) (2019R1C1C1006709, 2018R1A5A2025079, and 2020M3F7A1094093); a grantof the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Insti-tute (KHIDI), funded by the Ministry of Health &amp; Welfare, South Korea (KHIDIHI19C1015010020);"The Alchemist Project" through the Ministry of Trade, Industry and Energy (MOTIE, Korea)(20012443), and Severance Hospital Research fund for Clinical excellence (SHRC) (C-2020-0032and C-2020-0025) (H.S.K.); The Swedish Cancer Society Pancreatic Cancer Fellowship, the Lions In-ternational Postdoctoral fellowship and the Sweden-America stipend (L.B.); the Sa ̃ o Paulo ResearchFoundation (FAPESP) Sa ̃ o Paulo, Brazil (2017/07117-7) (G.C.T); the Swiss National Science Founda-tion (SFNS), Switzerland Postdoc Mobility grant (P2SKP3_174785 and P400PB_186791) (F.A.V.); andthe DoD PRCRP Horizon Award, United States (W81XWH-19-PRCRP-HA) (S.L).</p