VEGFR2-Mediated NFAT Activation Assay

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.