384 well white microplate

Quantification of intracellular cAMP was performed using the HTRF cAMP competitive immunoassay (Cisbio. France) according to the manufacturer’s instructions. Briefly, 48 h post-transfection, 5,000 WTT-CHO cells/well were distributed in a 384-well white microplate (Greiner) in the absence (basal) or in the presence of isoproterenol (ISO, 10 µM) for 30 min at 37 °C and in the presence of 0,5 mM IBMX to prevent phosphodiesterase-mediated cAMP degradation. When indicated, cells were pretreated with 200 mM sucrose for 1 h at 37 °C to prevent receptor internalization. After the addition of Cryptate-labeled cAMP (donor) and anti-cAMP-d2 (acceptor) for 1 h, the specific FRET signals were calculated by the fluorescence ratio of the acceptor and donor emission signal (665/620 nm) collected using a modified Infinite F500 (Tecan Group Ltd). Conversion of the HTRF ratio of each sample into cAMP concentrations was performed on the basis of a standard curve to determine the linear dynamic range of the assay.

Quantification of intracellular IP1 was performed using the HTRF (Homogeneous Time Resolved Fluorescence) IP1 competitive immunoassay (IP-One Tb kit. Cisbio. France) according to the manufacturer’s instructions. Briefly, 20,000 HEK293T/17 cells or 3.10⁶ platelets were distributed in a 384-well white microplate (Greiner) and incubated with the indicated molecules for 30 min or 2 h at 37 °C in the presence of 50 mM of LiCl to prevent IP1 degradation. After addition of d2-labeled IP1 (acceptor) and anti-IP1-Cryptate (donor) for 1 h, the specific FRET signals were calculated by the fluorescence ratio of the acceptor and donor emission signal (665/620 nm) collected using a modified Infinite F500 (Tecan Group Ltd). Conversion of the HTRF ratio of each sample into IP1 concentrations was performed on the basis of a standard curve to determine the linear dynamic range of the assay.