2.4. IP1 Measurement Assay

Briefly, the activation of GPR40 receptors in stable Chem1-GPR40 cells induced diacylglycerol (DAG) and inositol trisphosphate (IP3) production. The rapid degradation of IP3 was inhibited by lithium chloride (LiCl) addition in the assay. Cellular IP1 content accumulation was measured using the competitive immunoassay based HTRF^® IPone-Gq kit (Homogeneous Time Resolved FRET from Cisbio, Codolet, France). IP1 produced by GPR40 activation competes with an IP1 analog coupled to a d2 fluorophore (acceptor) for binding to an anti-IP1 monoclonal antibody labeled with Europium Cryptate (donor). Upon excitation at 330 nm, the Europium Cryptate emits light at 620 nm. When the IP1 analog binds to the anti-IP1 monoclonal antibody the donor and acceptor come in close proximity, and fluorescence resonance energy transfer (FRET) occurs, leading to signal emission at 665 nm from the donor. This signal is inversely proportional to the amount of phosphorylated substrate. The ratio between 665/620 was measured using a Synergy neo multimode reader (BioTek, Winooski, VT, USA) and results are expressed as Ratio (665_signal/620_signal x − 10,000). Chem1-GPR40 cells (10,000 per wells) were plated in a 384 Corning^® (#3657) in 50 μL culture medium for 24 h. Cells were then washed twice with 50 μL of assay buffer containing 146 mM NaCl, 10 mM HEPES, 5.5 mM glucose, 4.2 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂ and 50 mM LiC,l and cells were then starved for 30 min in 20 mL of assay buffer before treatment. A volume of 10 µL of a 3× concentration of each of the tested compounds was then prepared in assay buffer (1% DMSO final) and added to the cells for 90 min. The reaction was stopped by the addition of 6 mL of IP1 d2 and 6 mL of anti-IP1 in lysis buffer. After 24 h, the plate content was transferred to a white, low volume, 384-well plate Corning^® (#3824). After an excitation at 330 nm, emission signals at 620 nm and 665 nm were recorded, and a negative ratio (665_signal/620 _signal x − 10,000) was calculated.