Enrichment of Extracellular Vesicles from NCI-60 Cell Lines

Sixty cell lines from the National Cancer Institute (NCI-60) were acquired and cultured, as previously described [40 (link)]. MCF10a cells were grown using the Mammary Epithelial Cell Growth Medium BulletKit (Lonza, Basel, Switzerland, CC-3150) comprised of the basal medium MEBM supplemented with the provided aliquots of bovine pituitary extract (BPE), human recombinant epidermal growth factor (hEGF), insulin, and hydrocortisone. Instead of the GA-1000 aliquot provided in the kit, 100 ng/mL of cholera toxin was added to the medium, as recommended by the American Type Culture Collection (ATCC). At 90% confluence, the complete medium was aspirated and cells were washed with warm sterile phosphate buffered saline (PBS). To minimize contaminating proteins, cells were grown in BPE-free medium for another 48 h before EV enrichment.