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| | cAMP Assay |
| Description: | 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 μL of reduced serum medium containing 0.5% FBE. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 μl of culture medium containing 500 μM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 μM and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000× rpm for 10 minutes. 6. Aspirate the supernatant. 7. Add 100 μL of ETA assay buffer to each well and put the plate with the lid in a −80° C. freezer. Freeze the sample in the −80° C. for at least one hour. 8. Take the plate out from the −80° C. freezer and leave it at room temperature to thaw completely. 9. Centrifuge the plate at 1,000× rpm for 10 minutes. 10. Pick up 50 μl of supernatant from each well for cAMP level measurement, using an ELISA assay kit from Cayman chemical, Item #581001. 11. The data was analyzed and the EC50 for PGE2 and each test compound was calculated using GraphPad Prism 5. |
| Affinity data for this assay | |
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