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Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture\u2013conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles\/\n                    <jats:bold>\u03bc<\/jats:bold>\n                    g EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.\n                  <\/jats:p>","DOI":"10.1172\/jci.insight.137937","type":"journal-article","created":{"date-parts":[[2021,8,10]],"date-time":"2021-08-10T16:41:56Z","timestamp":1628613716000},"source":"Crossref","is-referenced-by-count":4,"title":["Optimizing extracellular vesicles\u2019 isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant"],"prefix":"10.1172","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2274-9891","authenticated-orcid":true,"given":"Sara","family":"Elgamal","sequence":"first","affiliation":[{"name":"Division of Hematology, Department of Internal Medicine, College of Medicine,"},{"name":"Comprehensive Cancer Center,"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emanuele","family":"Cocucci","sequence":"additional","affiliation":[{"name":"Comprehensive Cancer 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