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| | EVALUATION OF THE INHIBITION OF PAFR ACTIVATION |
| Description: | USING AN ENDPOINT ASSAY HEK293 cells overexpressing human PAFR (produced in-house) are seeded into a Poly-D-Lysine coated 384 well white cell culture microtiter assay plate with lid (15.000 cells per well). Subsequently, the plates are incubated over night at 37 °C/5% CO2. On the next day, cells are being washed and subsequently, various concentrations of the test compounds (compounds in 100% DMSO; final concentratrion of DMSO in wells is 1%) are added to the assay plate via Echo 555 acoustic liquid handler. Plates are then incubated with lid for 90 minutes at 37 °C/5% CO2. Thereafter, PAF ligand (Cayman Chemical Company, Item no.: 60900) is added at a final concentration of 11 nM. Plates are then incubated with lid for 60 minutes at 37 °C/5% CO2. Then, 5 µl per well of Anti-IP1-Antibody-Cryptate solution and 5 µL per well of IP1-d2 solution are added to all wells of the plate and the plate is incubated for another 60 minutes light protected at room temperature. The emissions at 620 nm and 665 nm (Excitation wavelength: 320 nm) are measured using an Envision Reader (PerkinElmer). |
| Affinity data for this assay | |
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