Transwell THP-1 migration assay

THP-1 were grown in RPMI-1640 (Thermo Fisher) medium supplemented with 5% vesicles-depleted foetal bovine serum (System Bioscience) and 1% Penicillin-Streptomycin-Amphotericin B (Lonza). The migration capacity of THP1 was determined using 8 μm pore polycarbonate filter transwell plates (HTS Transwell 96 well-permeable support, CORNING, USA). Briefly, the top of the transwell insert was seeded with 50 μL pre-starved THP1 in serum-free RPMI1640 medium (1 × 10⁶ cells/mL) and the lower chamber was filled with 100 μL serum-free RPMI1640 medium supplemented with either 0% FBS (negative control), a serial dilution of EV samples or 50 ng/mL recombinant human MCP1 (PeproTECH, Rocky Hill, CT, USA) as a positive control. For the EV samples, a serial dilution of size-based EV population (6.25 E07, 1.25 E08, 2.5 E08, 5E08 and 1 E09 particles/mL) or 2.5 E08 particles/mL of the immuno-depleted fractions in RPMI1640 medium supplemented with 0% FBS was placed directly into the lower chamber. Again the equally sized volumes of EV-depleted cell culture supernatant (sEV (-)) were used as a control. The number of cells that passed through the membrane was counted in the lower chambers using trypan blue 0.4% (Thermo Fisher Scientific) after overnight incubation (~16 h) at 37°C. The percentage of migrated cells was calculated for each condition in three independent experiments with three technical replicates (n = 3).