Envision imager

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).

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 24 hours. IP₁ accumulation was measured using Homogeneous Time-Resolved Fluorescence (HTRF) IP-ONE Tb Kit (Cisbio, Codolet, France) as per manufacturer’s instruction. The assay was perfomed using 10,000 cells/well into white, low volume 384-well plates (Greiner Bio-One) The HTRF signal was read on an Envision imager (Perkin-Elmer) after a 1 hour room temperature incubation.