Ip one kit

CHO-K1 cells were seeded in six-well plates and transfected with the indicated plasmids. Cells were then lifted using TrypLE Express (Thermo Fisher Scientific) and resuspended in 400 μl DMEM/F12. Cell suspension (7 μl/well) was transferred to a white opaque 384-well plate before adding agonist in stimulation buffer (included in kit). Cells were incubated for 1 h at 37°C, lysed and accumulated IP₁ was measured using the IP-One kit (Cisbio) following the manufacturer’s instructions. Readings were measured with a SpectraMax i3 plate reader (Molecular Devices, Sunnyvale, CA). Each condition was run in triplicate, and experiments were repeated independently at least three times.

The Cisbio IP-One kit was used according to the manufacturer’s instructions. HEK293 cells (ATCC CRL-1573) were seeded onto poly-L-Lysine-coated 384-well plates at 20,000 cells per well and transfected with 40 ng of DNA coding for the wild-type CysLT₂R or for the CysLT₂R mutants using the X-treme-Gene HP (Roche) agent. At 48 h post transfection, the media was removed and the cells were washed with fresh Hank’s Balanced Salt Solution. Cells were either stimulated directly with a range of LTD₄ concentrations (10⁻¹²–10⁻⁶ M) prepared in IP₁ stimulation buffer, or sequentially stimulated with a range of antagonist concentrations (10⁻¹¹–10⁻⁵ M), and LTD₄ concentrations corresponding to the EC₈₀ for each mutant. No LTD₄ degradation was observed by mass spectrometry (Supplementary Fig. 8). After equilibration for 30 min at 37 °C, the cells were lysed with IP₁-D2 and Ab-Crypt reagents in lysis buffer and then incubated for 1 h at RT. Fluorescence signal was recorded on a Tecan GENios Pro plate reader using an HTRF filter set (λ_ex 320 nm, λ_em 620 and 655 nm). Data were plotted using the three parameters EC₅₀/IC₅₀ fit in GraphPad Prism 7 (San Diego, CA) and represent the mean ± s.d. of at least two independent experiments performed in quadruplicate.

The same cells and controls were utilized as in the primary FLIPR assay. On the day of screening, cells were thawed and added to plates (3 μL/well, 1,500 cells/well) in growth media. This was followed immediately by pin-tool addition of test compounds and controls (positive control was 10 μM SR-01000435409; negative control was DMSO only) in 26 nL DMSO. The plates were incubated for 17 h at 37 °C, 5% CO₂ prior to addition of 3 μL of 1,000 nM GnRH in 2X stimulation buffer or stimulation buffer only (buffer provided in CisBio IP-One Kit). The plates were incubated for 2 h at 37 °C and 5% CO₂. 1.5 μL IP-One D2 (1:20 dilution—CisBio) and 1.5 μl IP-One Cryptate (1:20 dilution—CisBio) were added to each well and incubated for 1 h at room temperature in the dark. The plates were read on Viewlux (PerkinElmer Life Sciences) with excitation at 340 nM and emissions at 618 nM and 671 nM. A detailed outline of the confirmation assay can be found in Supplemental Table 2. The concentration responses of the control compounds are shown in Fig. 2.Figure 2

1536-well assay response to GnRHR controls. (A) Concentration–response of the controls when tested in the Fluo2 assay vs. (B) the IP-One TR-FRET assay. EC₅₀ values are shown in M. N = 16 replicates per concentration; error bars presented in SD.

Receptor function was determined by measuring intracellular inositol monophosphate concentration using the IP-one kit from Cis-bio according to the procedure described previously (see Supporting Information for details)^{21 (link)}. R 892 and WIN 64,338 hydrochloride (both from Tocris) were used as reference antagonists for B1R and B2R, respectively^{51 (link)}.