Inositol Phosphate 1 Assay

Accumulation of myo-inositol phosphate 1 (IP₁), a downstream metabolite of IP₃, was measured by using a IP-One homogeneous time-resolved fluorescence (HTRF) kit (catalog no. 62, IPAPEB; Cisbio, Bedford, MA). Functional coupling of the CB₁ receptor to G_q G protein leads to phospholipase Cβ activation and initiation of the inositol phosphate (IP) cascade. Accumulated IP₃ is quickly dephosphorylated to IP₂ and then IP₁. This assay takes advantage of the fact that accumulated IP₁ is protected from further degradation by the addition of lithium chloride and IP₁ levels can be easily quantified using a HTRF assay. HA-CB₁ HEK cells were detached from approximately 60% confluent plates/dish using versene. Cells (10 µl, 5000 cells) were resuspended in 1× stimulation buffer (containing lithium chloride, supplied with the kit) and were incubated for 1 hour at 37°C in 5% CO₂, humidified air and then transferred to a 384-well OptiPlate, followed by stimulation with drugs/compounds made in dimethylsulfoxide/ethanol as appropriate, for 10 minutes. Cells were then lysed with 5 µl IP₁-d₂ (made fresh in lysis buffer, supplied with the kit), followed by addition of 5 µl Ab-cryptate (made fresh in lysis buffer). Plates were incubated further for 90 minutes at room temperature and then read in HTRF mode on an Enspire plate reader. All cell-based assay experiments were performed in triplicate and were repeated at least two times, unless mentioned otherwise.