Mcf 7

Human BC cell lines MCF-7 and MDA-MB-231(the Cell Bank of Chinese Academy of Sciences, Shanghai, China), were incubated in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY, USA) including 10% fetal bovine serum (FBS), penicillin (1 × 10⁵ U/L) and streptomycin (100 mg/L) at 37 ℃ with 5% CO₂.
The EVs isolated from the serum of BC patients were incubated with PKH67 dyeing at 2–8 ℃ for 15–30 min. The supernatant was harvested after centrifugation, and samples were washed in PBS and resuspended. The suspension of 500 µL was detected on the flow cytometer at 490/502 nm. Then the stained EVs of 20 µL were respectively added into MCF-7 and MDA-MB-231 cells with 80% confluence. The entry of EVs into MCF-7 and MDA-MB-231 cells was observed under the confocal laser microscope (Olympus, Tokyo, Japan).

The MCF7 and MDA-MB-231 human BC cell lines were purchased by the American Type Culture Collection (ATCC). MCF7 and MDA-MB-231 cells were cultured in RPMI-1640 and DMEM respectively, supplemented with 10% fetal bovine serum (Euroclone, Milan, Italy), 1% glutamine and 1% penicillin/streptomycin at 37°C in 5% CO₂ humidified atmosphere. An amount of 20,000 BC cells were resuspended in medium serum-free and filtered through a 40 μm nylon mesh (Becton Dickinson, Franklin Lakes, NJ) before being suspended in 1.5 cm² low attachment wells (Becton Dickinson, Franklin Lakes, NJ) with 1 ml of medium serum-free, 1% penicillin/streptomycin. BC cells were incubated at 37°C in 5% CO₂ humidified atmosphere. MCF7 and MDA-MB-231 derived-MS were counted after 6 days of incubation as average number of cells per field of view with OLYMPUS CKX41 microscopy.

MCF-7 and ZR-75-1 cells were purchased from Bioresource Collection and Research Center (Food Industry Research and Development Institute, Taiwan) and grown in culture flasks (Nunc, Roskilde, Denmark) containing RPMI1640, with 10% heat-inactivated fetal bovine serum (FBS) and streptomycin sulfate, in a 5% CO₂ atmosphere at 37 °C until cells were sub-confluent. After incubation, morphological comparison between MCF-7 and ZR-75-1 cells was observed by using the Olympus microscope (IX71) at 20× fitted with an ocular grid.