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).
Bibliographical noteFunding Information:
We thank the Electron Microscopy & Histology services of the Weill Cornell Medicine Microscopy & Image Analysis Core for TEM with NIH , United States Shared Instrumentation Grant ( S10RR027699 ) and Medical Illustration & Design, part of the Medical Research Support Services of Yonsei University College of Medicine , South Korea, for all artistic support related to this work. We are grateful for the great AF4 technical support from Wyatt Technology. The authors gratefully acknowledge support from: the 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 , United States 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’s Consortium (D.L., T.M.T.); Alex’s Lemonade Stand Foundation , United Sates (D.L.); the Breast Cancer Research Foundation , United States (D.L.); the Feldstein Medical Foundation (D.L.); the Tortolani Foundation (D.L.); the Clinical & Translational Science Center (D.L., H.Z.); the Mary Kay Ash Charitable Foundation (D.L., I.M.); the Malcolm Hewitt Weiner Foundation (D.L.) ; the Manning Foundation (DL., A.H.) ; the Daniel P. and Nancy C. Paduano Family Foundation (D.L) ; the James Paduano Foundation (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 Postdoctoral 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 grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & 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-2020-0032 and C-2020-0025 ) (H.S.K.); The Swedish Cancer Society Pancreatic Cancer Fellowship, the Lions International Postdoctoral fellowship and the Sweden-America stipend (L.B.); the São Paulo Research Foundation (FAPESP) São Paulo, Brazil ( 2017/07117-7 ) (G.C.T); the Swiss National Science Foundation (SFNS), Switzerland Postdoc Mobility grant ( P2SKP3_174785 and P400PB_186791 ) (F.A.V.); and the DoD PRCRP Horizon Award, United States ( W81XWH-19-PRCRP-HA ) (S.L).
© 2020 The Author(s)
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)