T 225 flasks

Pancreatic carcinoma cell line, Panc-1 cells, containing KRAS G12D mutation and breast carcinoma cell line, MDA-MB-231 cells, without KRAS mutation were purchased from the American Type Culture Collection. All cells passed testing for mycoplasma contamination and were maintained in phenol-red-free-DMEM medium (Corning) supplemented with 10% (v/v) FBS, 100 units ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C. Panc-1 and MDA-MB-231 cells were grown in three T225 flasks (Falcon) for two to three days until they reached a confluency of 80%. Next, cells were cultured in medium without FBS for 48 h. The medium was collected and centrifuged at 300 g for 5 min followed by 16,500 g for 20 min at 4°C. Plasma was then collected and filtered using a 0.22 μm pore filter. A total of 108 ml of medium was collected and continuously ultracentrifuged at 100,000 g at 4°C for 2 h. The EV pellets were suspended in 200 μl of PBS. Samples were incubated with 10 μl of DNase I (1 unit μl⁻¹; Life Technologies) or 5 μg ml⁻¹ RNase at 37°C for 2 h. The supernatant was collected and stored at −80°C.