A tube formation assay was utilized to model the reorganization and tube forming processes of angiogenesis. The assay was performed in 96‐well plates precoated with Matrigel reduced growth factor basement membrane extract (Corning, Corning, NY, USA). Prior to the assay, 96‐well plates and pipette tips were chilled at −20°C and the Matrigel extract was thawed in ice at 4°C overnight. Each well was coated with 50 μl of Matrigel and incubated for 30 min at 37°C. During incubation, HUVECs were trypsinized and counted in serum‐free DMEM (Sigma‐Aldrich, St Louis, MO, USA). Cells were seeded onto the Matrigel‐coated wells at 12,000 cells per well and treated with EV in serum‐free DMEM. All treatments were done in duplicate. Cells were imaged after 6 h at ×25 magnification on a Leica DMi6000 microscope. Tube formation was measured using the angiogenesis analyser tool on ImageJ (NIH) as previously described (Carpentier et al., 2020 (link)). Total tubule length and number of tubes per field of view are reported.
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