IP1 Accumulation Assay.

Accumulation of IP1, a downstream metabolite of IP3, was measured by using IP-One HTRFkit (cat. no. 62, IPAPEB; Cisbio, Bedford, MA). Functional coupling of CB2 receptor to G_q G protein leads to phospholipase Cβ (PLC) activation and initiation of the IP hydrolysis cascade. Accumulated IP3 is quickly dephosphorylated to IP2 and then to IP1. This assay takes advantage of the fact that accumulated IP1 is protected from further dephosphorylation by the addition of lithium chloride, and IP1 levels can be easily quantified using an homogeneous time-resolved fluorescence (HTRF) assay. HEK mCB2 cells were detached from ∼50% confluent plates 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, 5% CO₂, and humidified air and then transferred to a 384-optiplate, followed by stimulation with drugs/compounds made in DMSO/ethanol as appropriate, for defined time points. Cells were then lysed with 5 µl of IP1-d2 dye (made fresh in lysis buffer, supplied with the kit), followed by the addition of 5 µl Ab-Cryptate dye (made fresh in lysis buffer). Plates were incubated further for 60 minutes at room temperature and then read in HTRF mode on an Enspire plate reader. All cell-based assay experiments were performed in triplicate unless otherwise stated.