| Assay Method Information | |
| | hEP4 cAMP Assay |
| Description: | The BT549 cells (NCI/No. 0507282) are detached from culture dishes with a cell dissociation buffer (Invitrogen, 13151-014), and collected in growing medium (GM: RPMI1640 (Invitrogen 21875)/10% FCS, 1% Penicilin/streptomycin). Cells are counted washed and resuspended in assay buffer (AB; HBSS, 20 mM HEPES, 0.2% BSA; 2 mM IBMX). 4'000 cells in 5 μl of AB are seeded per well of a small volume 384 well plate (black with flat bottom, Greiner 784076).Stock solutions of test compounds are made at a concentration of 10 mM in DMSO, and serially diluted in DMSO to concentrations required for inhibition dose response curves (tested concentration range 30 μM-0.4 nM; 30 μM-0.015 nM or 1 μM-0.01 nM).PGE2 (Cayman 14010, stock solution: 6 μM in DMSO) is used as agonist at 6 nM final concentration, corresponding to EC80.Two point five microliters of diluted compounds are transferred into the assay plate. Plate is pre-incubated 45 minutes at RT. Subsequently, 2.5 microliters of PGE2 (final conc. 6 nM) are transferred into the assay plate. Plate is incubated 30 minutes at RT. Five μl of each donor (anti-cAMP cryptate) and acceptor (cAMP-d2) are added and the plate is incubated another hour at RT in the dark and then read using a BMG LABTECH PHERAstar reader (Excitation: 337 nm, Emission: 620 and 665 nm).The obtained Delta F (fluorescence) values (665 nm/620 nM) are converted into % cAMP values using the measurements of the cAMP calibrator provided in the kit. For each compound concentration the percentage of cAMP compared to DMSO control value as average ±STDEV (each concentration is measured in duplicate) is calculated. |
| Affinity data for this assay | |
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