4.2. Purification of leukaemia EVs from THP1 cells

THP1 cells were obtained from the American Type Culture Collection (ATCC, USA) and maintained in RPMI (Thermo Fisher Scientific, USA), containing 10% foetal bovine serum (Biosera, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific). To make EV‐free FBS, EVs were removed from FBS using ultracentrifugation at 110,000 × g for 18 h at 4˚C. THP1 cells were cultured at 10⁶ cells/ml in the above medium with EV‐free FBS and 0.2 μM calcium ionophore for 48 h. Culture supernatants were collected from 5 flasks of treated THP1 cells. Cells and debris were separated by differential centrifugation at 300 × g for 10 min, 400 × g for 15 min, 900 × g for 15 min at 4˚C. The supernatant was further passed through a 0.45 μm filter, placed on top of 2 ml frozen 60% sucrose, and enriched by ultracentrifugation with a SW32 rotor at 100,000 × g for 90 min at 4˚C. THP‐1 derived EVs were collected from the interface and diluted 1:1 in cold PBS, and layered above 2 ml frozen 60% sucrose cushion in a SW41 rotor and centrifuged at 100,000 × g for 12 h at 4˚C (Beckman Coulter, USA) with reduced braking speed. 500 μl EVs were collected from the interface and added to a qEV SEC column (Izon, New Zealand). A total of 500 μl eluate was collected in each fraction. The concentration of EVs and protein were measured in 30 fractions using a Nanosight analyzer and bicinchoninic acid (BCA) assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific). For ligation, the EVs from fraction 7 to 11 were combined and concentrated using centrifugation at 15,000 × g for 20 min in an Amicon‐15 filter with 100 kDa cut‐off.