Mammary epithelial cell growth medium singlequots kit

Human BC cell lines MCF7 and MDA‐MB‐231 and normal human breast epithelial telomerase immortalized cells 184‐h TERT were cultivated to isolate EVs. Breast cancer cells were cultured in DMEM supplemented with 10% FBS (Gibco/Thermo Fisher Scientific) and 1% penicillin/streptomycin (Corning/Mediatech) in accordance with the suppliers’ recommendations. After the cells were washed with PBS, the culture medium was replaced with FBS‐free advanced DMEM and collected after 48 hours of incubation for EV isolation. 184‐h TERT cells were cultivated in serum‐free Mammary Epithelial Basal Medium (Lonza) supplemented with Mammary Epithelial Cell Growth Medium SingleQuots Kit (Lonza) in accordance with the supplier’s recommendations. After the cells were washed with PBS, the culture medium was collected after 48 hours of incubation. Before isolation of EVs, collected cell supernatant was centrifuged at 2000 g for 30 minutes at 4°C and at 12 000 g for 30 minutes at 4°C to remove dead cells and cellular debris and large vesicles were discarded. After centrifugation, the supernatant was filtered through a 0.22‐μm pore filter. All cells were incubated in a humidified chamber at 37°C and 5% CO₂.