HTRF Assay for IP1 Accumulation

The determination of IP₁ accumulation was performed in a 96-well microplate seeded with transfected HEK293FT cells (500 ng/well of a 6-well plate) using the IP-One HTRF assay (CisBio Bioassays) (18 (link), 27 (link)). To construct concentration-response curves, cells expressing either wild-type V2R/Nluc or L312S V2R/Nluc were incubated for 30 minutes at 37°C in the stimulation buffer (10mM HEPES [pH 7.4], 1mM CaCl₂, 0.5mM MgCl₂, 4mM KCl, 146mM NaCl, 5.5mM glucose, and 50mM LiCl) with or without 1pM–10μM AVP or carbachol (2mM) as an internal control. Cells were then lysed using the conjugate-lysis buffer mixed with the d2-labeled IP₁ analog and the terbium cryptate-labeled anti-IP₁ antibody according to the manufacturer's instructions. The assay was incubated for 1 hour at room temperature before fluorescence signals were measured at 620 and 665 nm, 50 microseconds after excitation at 337 nm using a CLARIOstar multilabel plate reader.