NFAT-Luciferase reporter assay

NB Nisha Bhudia
SD Sapna Desai
NK Natalie King
NA Nicolas Ancellin
DG Didier Grillot
AB Ashley A. Barnes
SD Simon J. Dowell
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HEK 293 T cells were seeded into T75 flasks at 90,000 cells/cm2 and incubated at 37 °C and 5% CO2 overnight. The following day, cells were transiently transfected for 48 hours in total; for the last 24 hours, cells were plated at 10,000 cells/well into sterile, white, 384-well plates (Nunc/Thermo Fisher Scientific) containing 5–10 mg/ml of EMR2 antibody or 25 μM AngII (human Angiotensin II, Sigma-Aldrich) in phenol-red free OptiMEM1 medium (Gibco/Thermo Fisher). Luminescence was measured using the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA) as per manufacturer’s instructions. The Luminescent signal was read on a EnVision imager (Perkin-Elmer).

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