Htrf camp assay kit

IP formation was quantified with HTRF (Homogeneous Time-Resolved Fluorescence) based “Cisbio IP-One Tb” (Cisbio, Bagnols-sur-Cèze, France) assay kit, following manufacturer’s instructions. EndoC-βH2 cell suspensions (5 × 10⁴ cells) were treated in 384-well plate (16 μl volume) with modified stimulation buffer (10 mM Hepes, 10 mM MES, 1 mM CaCl2, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM LiCl) at pH 7.4 or 6.4 for 60 min at 37 °C. Where indicated, cells were pretreated with YM-254890, a selective Gα_q/11 inhibitor27 for 30 min prior to incubation with the IP stimulation buffer and maintained throughout the IP determination. IP measurements were performed in triplicates and experiments were repeated at least three times. Samples were read on a TECAN Infinite F500 (Tecan Group, Ltd., Männedorf, Switzerland) with excitation at 320 nm and emission at both 620 nm and 665 nm.
cAMP activity was measured using a cAMP-HTRF assay kit (Cisbio) following manufacturer’s instructions. EndoC-βH2 cells (5 × 10³) were treated in 384-well plate (12 μl final volume) with stimulation buffer (PBS containing 10 mM of each HEPES, MES, and 0.5 mM IBMX, at pH 7.4 or 6.4) for 30 min at room temperature. Cells were lysed using kit lysis buffer and cAMP was then measured in 384 well plates (HTRF) with TECAN Infinite F500.