Primary human foreskin fibroblasts

Primary human foreskin fibroblasts (Promocell) were cultured until confluent in 48-well plates in Q333 fibroblast growth media (PAA Laboratories, Pasching, Austria). 7500 endothelial cells were then seeded per well onto the fibroblasts monolayer in a 1:1 mixture of Q333 and ECGM and left to acclimatize for 24 h. Media was then aspirated and replaced with fresh ECGM±VEGF-A isoform (1.25 nM) as desired; media was replaced every 2-3 days for seven days. Co-cultures were then fixed in 200 µl 10% (v/v) formalin for 20 min and blocked in 5% (w/v) BSA for 30 min at room temperature. Co-cultures were then incubated with 1 µg/ml mouse anti-human PECAM-1 (CD31; Santa Cruz, USA) overnight at 4°C. Cells were washed three times with PBS before incubation with an anti-mouse Alexa Fluor 594 conjugate (Invitrogen) for 3 h at room temperature. Wells were then washed three times with PBS. Endothelial tubules were visualized by immunofluorescence microscopy using an EVOS-fl inverted digital microscope (Life Technologies, Paisley, UK). Five random fields were imaged per well. Both the number of branch points and the total tubule length was then quantified from each photographic field using the open source software AngioQuant (www.cs.tut.fi/sgn/csb/angioquant) and values averaged. For a more detailed method please see Fearnley et al., (2014b (link)).