| Assay Method Information | |
| | Calcium Flux Assays (hB1 IC50 IMR90) |
| Description: | Notably, Compound Examples are tested in the FLIPR assays in the presence (hB1 free IC50) of 0.1% BSA in assay buffer, in order to assess the potency shifts due to serum protein binding of Compound Examples. The effect of BSA on the potency of endothelin receptor antagonists have been described in the prior art (Wu-Wong, J. R. et al. (1997), JPET 281: 791-798). The teaching can be applied in analogy to testing the potency of Bradykinin B1 receptor antagonist in the FLIPR assays. For the calcium flux assay, 80% confluent cells are detached from the culture vessels with Versene (Gibco), and seeded into 384-well plates (Cell binding Surface; Corning, N.Y.; #3683) at a density of 15,000 cells per well. Cells are seeded in a volume of 50 μL in medium without antibiotics and incubated overnight in a humidified atmosphere with 5% CO2 at 37° C. The following day, the medium is replaced with 20 μL of 5 μM Fluo-4AM dye (Molecular Probes) in assay buffer (2.5 mM probenicid, 1 mg/mL pluronic acid, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl, 1 mM MgCl2, 10 mM HEPES, 5.6 mM glucose, and 0.05% gelatine, pH 7.4), which contains or lacks 0.1% BSA for determination of compound potency units as hB1 IC50 or hB1 free IC50, respectively. The calcium indicator loaded cells are incubated at 37° C. for 2 hrs. Extracellular dye is then removed and each well is filled with 45 μL of assay buffer. Cell plates are kept in dark until used. Compound examples are assayed at 8 concentrations in triplicate. Serial 10-fold dilutions in 100% DMSO are made at a 100-times higher concentration than the final concentration, and then diluted 1:10 in assay buffer. 5 μL of each diluted compound is added to the well of cell plates (yielding final concentration with 1% DMSO), and incubated for 30 min at 28° C. before the addition of Bradykinin B1 receptor agonist on the FLIPR instrument. Agonist plates contain the agonist Lys-(Des-Arg)-Bradykinin (Bachem, Brackley) at 3.5×EC90 in assay buffer with 1% DMSO. The addition of agonist 20 μl per well to the assay plate is carried out on the FLIPR instrument while continuously monitoring Ca2+-dependent fluorescence at 538 nm. A peptide antagonist Lys-(Des-Arg-Leu)-Bradykinin (Bachem, Brackley) at 20 □M is used to determine the full inhibition as control. Peak fluorescence is used to determine the response to agonist obtained at each concentration of Compound Examples by the following equation: % Response=100*(RFU(compound)−RFU(control))/(RFU(DMSO)−RFU(control)) RFU means relative fluorescence units. Control means full inhibition by the peptide antagonist Lys-(Des-Arg-Leu)-Bradykinin at 20 □M.The response values are plotted against the logarithm of the compound concentrations. The Compound Examples are tested in triplicates per plate and mean values are plotted in Excel XLfit to determine IC50 values, percentage of maximal inhibition and the Hill slopes. |
| Affinity data for this assay | |
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