EC migration was performed on confluent monolayers in gelatin-coated 96-well cell culture plates. A wound of approximately 300 µm wide was made using a guided 96-well pin tool (Peira, Turnhout, Belgium). Wells were washed to remove cell debris and the medium was added. Images were captured with a Leica DMI3000 microscope (Leica, Rijswijk, The Netherlands) with Universal Grab 6.3 software (DCILabs, Keerbergen, Belgium), at time points T = 0 h to T = 8 h61 (link). Where indicated, wound closure (μm2) was expressed as a percentage of control wells.
EC spheroids were created using the hanging drop technology72 (link). Briefly, 1000 HUVEC were resuspended in 25 μl aliquots containing medium supplemented with 20% methocel (Sigma-Aldrich), and drops were incubated on the inverted lid of a PBS-filled petri dish. Twenty-four hours later, the drops were gently harvested and embedded in type I bovine collagen gel (2 mg/ml, Advanced BioMatrix), at approximately 20 spheroids per well in 8-well microscope slides (Ibidi). After solidification, medium and compounds were added on top of the gel. Quantification of sprouting was performed using a semi-automatic ImageJ-based macro73 (link) on 5–25 spheroids per condition. Cells were treated as indicated.
EC spheroids were created using the hanging drop technology72 (link). Briefly, 1000 HUVEC were resuspended in 25 μl aliquots containing medium supplemented with 20% methocel (Sigma-Aldrich), and drops were incubated on the inverted lid of a PBS-filled petri dish. Twenty-four hours later, the drops were gently harvested and embedded in type I bovine collagen gel (2 mg/ml, Advanced BioMatrix), at approximately 20 spheroids per well in 8-well microscope slides (Ibidi). After solidification, medium and compounds were added on top of the gel. Quantification of sprouting was performed using a semi-automatic ImageJ-based macro73 (link) on 5–25 spheroids per condition. Cells were treated as indicated.