| Assay Method Information | |
| | HIF-1alpha transactivation assays |
| Description: | HaCaT-EPO-Luc cells were maintained at 37° C. in a humidified atmosphere containing 5% CO2 in DMEM supplemented with 10% fetal calf serum (FBS), and 1% (v/v) penicillin/streptomycin. The cells (1×105/well in 24-well plates) were seeded the day before the assay and then stimulated with increasing concentrations of either Cannabidiol (CBD), VCE-004 or compounds II to X. After six hours of stimulation the cells were lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCi2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min at RT in a horizontal shake. Luciferase activity was measured in the cell lysates as indicated for NIH3T3-EPO-Luc cells. The RLUs are calculated and the EC50 and IRA (Intrinsic relative activity) values in both cell lines were determined relative to 150 μM deferoxamine (DFX) using the following equation: IRA coefficient=(EC50-DFX×Emax)/(EC50×Emax-DFX), where EC50 and Emax denote EC50 and Emax of the agonist, and EC50-DFX and Emax-DFX denote EC50 and Emax values of the standard agonist DFX. |
| Affinity data for this assay | |
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