Ip one htrf

Determination of G protein–mediated signaling by human α_2AAR, murine α_2AAR, and human α_2BAR was performed applying an IP accumulation assay (IP-One HTRF, Cisbio, Codolet, France) according to the manufacturer’s protocol and in analogy to previously described protocols (77 (link), 78 (link)). In brief, HEK 293T cells were cotransfected with the cDNA for a receptor and the hybrid G protein Gα_qi (Gα_q protein with the last five amino acids at the C terminus replaced by the corresponding sequence of Gα_i) (gift from The J. David Gladstone Institutes, San Francisco, CA), respectively, in a ratio of 1:2. After 1 day, cells were transferred into 384-well microplates (Greiner) and incubated for further 24 hours. On the day of the experiment, cells were incubated with test compounds for 90 min (α_2AAR) or 120 min (α_2BAR), and accumulation of second messenger was stopped by adding detection reagents (IP1-d2 conjugate and Anti-IP1cryptate TB conjugate). After 60 min, TR-FRET was monitored with a Clariostar plate reader. FRET-signals were normalized to buffer (0%) and the maximum effect of norepinephrine (100%). Three to nine (murine α_2AAR, α_2BAR) or 4 to 11 repeats (human α_2AAR), respectively, were performed for each test compound all done in duplicate.