Reluc2p

HEK293T cells stably expressing both wild type VEGFR2 and the Firefly luciferase reporter gene ReLuc2P (Promega Corporation, USA) inserted downstream of the NFAT promoter were used to monitor NFAT-induced gene transcription following VEGFR2 activation (Carter et al., 2015 (link)). On the day of experimentation, cells grown to 95-100% confluency were plated in white-sided 96 well plates (Greiner Bio-One, 655089) at 44,000 cells/well, and incubated for 1 hour in 100μl/well serum free DMEM/0.1% BSA (37°C/5% CO₂). Cells were stimulated in duplicate wells with increasing concentrations of VEGF₁₂₁a-TMR, VEGF₁₆₅b-TMR or equivalent unlabelled VEGF isoforms (synthesised in an identical manner to the fluorescent variant), then incubated for 5 hours at 37°C/5% CO₂. ONE-Glo Luciferase reagent (Promega Corporation, USA) was then added at 100μl/well and luminescence was measured using a TopCount platereader (Perkin Elmer, UK) following a 5 minute delay allowing reagent to react with luciferase and background luminescence to subside.

HEK293T cells stably expressed HaloTag-VEGFR2 or NanoLuc-VEGFR2, as well as ReLuc2P, a Firefly luciferase reporter gene inserted downstream of the NFAT reporter to monitor NFAT-induced gene transcription (Promega Corporation, USA). Cells were grown to 70-80% confluency and seeded in DMEM containing 10% Fetal Calf Serum at 25,000 cells per well in white 96-well plates (Greiner Bio-One, 655089) pre-coated with poly-D-lysine (0.1mg/ml in PBS). Following 24 h of cell growth at 37°C/5% CO₂, plating medium was replaced with serum free DMEM for a further 24 h. Cells were stimulated in duplicate wells with increasing concentrations of VEGF₁₆₅a (R&D Systems) or vehicle (serum free DMEM containing 0.1% BSA) for 5 h at 37°C/5% CO₂. Assay medium was then replaced with 50μl/well serum free DMEM and 50μl/well One-Glo Luciferase reagent (Promega Corporation, USA) and equilibrated for 5 min to enable the reagent to react with luciferase and allow background luminescence to subside. Luminescence was then measured by a TopCount platereader (Perkin Elmer, UK). Data were normalised to vehicle (0%) and response to 10nM VEGF₁₆₅a (100%) in each experiment and expressed as mean ± SEM. (5 independent experiments). Potency (EC₅₀) values were derived as described previously (Kilpatrick et al., 2017 (link)).