Ip one gq assay kit

Flag-tagged wild-type and mutant FPR2s were cloned into the expression vector pTT5 (Invitrogen) and expressed in HEK293 cells (Invitrogen) along with the chimeric Gα protein Gα_Δ6qi4myr at the ratio of plasmids of 1:2 (w/w). Cells were routinely tested for mycoplasma contamination. Cells were harvested 48 h post transfection. Cell-surface expression of the receptor was measured by mixing 10 μl cells and 15 μl Monoclonal ANTI-FLAG M2-FITC antibody (Sigma, F4049; 1:100 diluted by TBS supplemented with 4% BSA). After 20 min, the fluorescence signal on the cell surface was measured by a FCM (flow cytometry) reader (Millipore).
IP1 accumulation was measured using an IP-One Gq assay kit (Cisbio Bioassays, 62IPAPEB) following the manufacturer’s instruction. In brief, the cells were plated in 384-well plates (20,000 cells per well) and treated with different concentrations of WKYMVm (1 pM–10 μM) or fMLFK (1 nM–1 mM) diluted in stimulation buffer at 37 °C for 90 min. Then 3 μl cryptate-labeled anti-IP1 monoclonal antibody and 3 μl d2-labeled IP1, which were pre-diluted in Lysis Buffer (1:20), were added to the wells, and incubated at room temperature for 1 h. Plates were read in a Synergy^TM H1 Operator (BioTek) with excitation at 330 nm and emission at 620 and 665 nm. The IP1 production was calculated according to a standard dose–response curve. Data were analyzed using Prism 7.0.

The IP‐one Gq assay kit (Cisbio) was used to quantify accumulated myo‐inositol‐1‐phosphate (IP₁), a by‐product of IP₃ produced after receptor‐mediated Gαq activation in Cos7 and HEK‐293S cells. Briefly, culture media was removed and replaced with DMEM containing 0.1% BSA for 30 minutes at 37°C. Cells were then incubated with media containing 50 mmol/L lithium chloride (LiCl) for an additional 0‐120 minutes (time courses) or 90 minutes at 37°C in the presence or absence of an antagonist. Agonist concentrations of 1 pmol/L to 10 µmol/L were used for agonist only assays or 10 pmol/L to 10 µmol/L concentrations for antagonist experiments. After cell stimulation, 14 µL of stimulation buffer was added to each well. Three microlitres of each detection antibody were added in turn to the plate and incubated at room temperature for 1 hour. Fifteen microliters of sample was then transferred to a 384‐well optiplate and read by an Envision plate reader (PerkinElmer). IP₁ concentrations were calculated from a standard curve generated in duplicate.

HEK293 cells were grown in 25 cm² culture flask and co-transfected with 800 ng plasmid encoding the Gα_Δ6qi4myr protein and 3200 ng plasmid encoding the Y₂R fused C-terminally to eYFP applying Metafectene® Pro (Biontex Laboratories GmbH) according to the manufacturer’s protocol. One-day post transfection, cells were seeded (75,000 cells/well) into white poly-D-lysine covert 96-well plates (Greiner Bio-one) and incubated overnight at 37 °C. Receptor activation studies were performed by using the IP-one Gq assay kit (Cis-Bio). Prior to detection of IP- species; cells were either stimulated with buffer, 100 nM or 1 μM NPY for 60 min and washed with acidic wash buffer and HBSS. For recycling, cells were incubated for 60 min with assay buffer, supplemented with 100 μg/ml CHX (Merck/Calbiochem®). After recovery, the recycling medium was removed and 30 μl stimulation solution containing NPY in the concentration range of 10^− 13 M to 10^− 7 M and LiCl (inhibition of IP-species degradation) was added and cells were incubated for 1.5 h at 37 °C. Stimulation was stopped by adding lysis buffer supplied with antibody 1 and antibody 2 according to the manufacturer’s protocol. After 60 min of incubation at room temperature, the emission at 620 nm and 665 nm was measured and the ratio (acceptor 665 nm/donor 620 nm) was calculated.