IP1 Accumulation Assay with HTRF

The determination of IP₁ accumulation was performed using the IP-One HTRF assay (CisBio Bioassays, Bagnol sur Ceze, France), as described previously [45 (link)]. Briefly, cells were transfected and seeded into white 96-well plates. 48 h post-transfection cell media was replaced with 50 μl stimulation buffer containing agonists as indicated. After a 30 min incubation at 37°C, 5% CO₂, cells were lysed with 12.5 μl of the supplied conjugate-lysis buffer containing d2-labeled IP₁. This was immediately followed by addition of 12.5 μl of conjugate-lysis buffer containing terbium cryptate-labeled anti-IP₁ antibody. Following a 1 h incubation at room temperature, fluorescence was measured at 620 and 665 nm 50 μs after excitation at 337 nm using an EnVision 2102 plate reader (PerkinElmer).