Inositol Phosphate one (IP-one) Homogenous Time Resolved Fluorescence (HTRF) assay

Cellular IP₁ levels were measured using an IP-one HTRF assay kit (Cisbio)¹³. Briefly, subconfluent cells were detached from the cell culture dish by trypsinisation and resuspended in the appropriate volume of the assay stimulation buffer (10 mM HEPES, 1 mM CaCl_2, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM LiCl, pH 7.4) warmed to 37 °C, to achieve a concentration of 6 × 10⁶ cells/ml. The cell suspension was added to a white half-volume 384 well plate (Greiner) along with the compound to be tested and incubated at 37 °C for 2 h. IP-one lysis buffer containing 5% IP-one-d₂ conjugate was added to the appropriate wells, followed by 5% anti-IP-one cryptate Tb conjugate. Samples were incubated in the dark for 1 h at room temperature. The plate was read on BMG labtech plate reader with an excitation at 340 nm and emission at 615 nm and 665 nm respectively. The fluorescence resonance energy transfer (FRET) ratios (665 nm/615 nm) were converted to IP₁ concentrations by interpolating values from an IP₁ standard curve.