| Assay Method Information | |
| | MRGPRX2 Activity Assay |
| Description: | FlpIn TRex-MRGPRX2-HEK293 cells (herein TRex-MRPGRX2-HEK) stably transfected to express human MRGPRX2 were maintained at 37oC with 5% CO2 and grown in DMEM supplemented with 10% FBS, 1% L-Glutamate, 1% penicillin/streptomycin, 100ug/mL Hygromycin B, and 15 µg/mL Blasticidin. GOI (MRGPRX2) expression was induced with 100ng/mL of Doxycycline 18-24 hours prior to experiment, in flask. On the day of experiment, cells were lifted with TripLE Express and seeded in 384-well white walled opaque bottomed, assay plates, at 8,000 cells per well in 5 µL freshly made assay buffer (HBSS, 20 mM HEPES, 0.1% BSA and 10 mM LiCl). Compounds solubilized in 100% DMSO at 30 mM were serially diluted (1:4) first in 100% DMSO, and an intermediate plate of assay buffer was used to establish a final top concentration of 30 µM (in assay plate). Final DMSO concentration was kept constant at 1% across assay plates. Cortistatin-14, at 30 mM stock concentration was solubilized in HBSS + 0.1% BSA, stored at -20°C in 5 µL aliquots and discarded after one freeze-thaw cycle. Final concentration of agonist used in antagonist mode assay was 1 ^M, estimated EC80 (EC50 of Cortistatin-14 ~ 2.5e-7 M). Antagonist equilibrium was established with 30 minutes preincubation, followed by no wash, direct addition, and stimulation of agonist for 1 hour at 37oC / 5% CO2. IP-1 standards and HTRF detection reagents were added according to the IP-one Gq kit protocol purchased from CisBio (part number 62IPAPEJ). The plate was read on an EnVision XCite plate reader. The HTRF ratio was then calculated from raw data (channel1/channel2 ×10,000) and analyzed using either GraphPad Prism or a custom Python script to calculate IC50 using Log[Antagonist] vs. response (4 parameter) nonlinear regression. |
| Affinity data for this assay | |
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