Mouse anti human pecam 1 cd31

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)).

Primary human foreskin fibroblasts (Promocell) were cultured in 48-well plates until confluent in complete DMEM containing 10% FCS, 1% non-essential amino acids and 1% sodium pyruvate (Life Technologies). 6500 HUVECs were then seeded onto the fibroblast monolayer in a 1:1 mixture of complete DMEM containing 10% (v/v) FCS, non-essential amino acids, sodium pyruvate and ECGM and left to adhere for 24 h. Culture media was then removed and replaced with fresh ECGM ±VEGF-A as desired; media was replaced every 2–3 days for 7 days. Co-cultures were then fixed and blocked prior to overnight incubation with 1 µg/ml mouse anti-human PECAM-1 (CD31; Santa Cruz Biotechnology) at room temperature. Co-cultures were washed three times with PBS before incubation with 1 µg/ml donkey anti-mouse Alexaflour594-conjugate (Invitrogen) for 2–3 h at room temperature. Endothelial tubules were then visualised by immunofluorescence microscopy using an EVOS-fl inverted digital microscope (Life Technologies). Three 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. A more detailed methodology is available elsewhere (Fearnley et al., 2014b (link)).