| Assay Method Information | |
| | Functional Ca2+ Mobilisation Assay |
| Description: | Ca2+ ions are usually kept at nanomolar levels in the cytosol of cells, and act in a number of signal transduction pathways as second messengers. Many GPCRs including neurotensin receptor couple to induce calcium ion signaling, and many primary cellular assays employ measurement of intracellular calcium ion concentration as a functional readout of GPCR activation. Changes in calcium ion concentration in standard assay protocols can be readily detected with fluorescent dyes that emit light when changes in intracellular Ca2+ ion concentration occur. Given the transient nature of these responses, they are often read with instrumentation that has inject and read capability. This example shows that compounds of the present invention do not have any agonistic activity on NTR1-expressing cells. Furthermore, this example shows that conjugates of the present invention bind to NTR1 and inhibit the activity of an additionally present NTR1 agonist.HT29 or NTR1-expressing HEK293 cells were trypsinized and seeded into black flat clear-bottom 96-well plates (Corning, Amsterdam, The Netherlands) at 6×105 cells per well. After 24 h incubation at 37° C. and 5% CO2, cells were washed twice with wash buffer (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl2, 10 mM Glucose, pH 7.4) and loaded with 100 μl of Ca5 dye (Molecular Devices, Biberach, Germany) for 1 h at 37° C. and 5% CO2. For agonist assays, serial dilutions of agonistic substances were added to the cells loaded with dye and the change of the fluorescent signal was recorded continually for approx. 90 s using a FlexStation II (Molecular Devices, Biberach, Germany). Addition of wash buffer served as a control. Thus, EC50 concentrations for each conjugate were computed and provided a measure for the potency of the substance. For antagonist assays, cells loaded with 100 μl of Ca5-dye were pre-incubated with serial dilutions of antagonistic substances for 30 min, before the EC80-concentration of agonist was added to the cells and the change of the fluorescent signal was recorded continually for approx. 90 s. Thus, IC50 concentrations were computed for each conjugate and provided a measure for the inhibitory activity of the conjugates at the NTR1.The intrinsic fluorescence of fluorescein-containing conjugate (19) interfered with the Ca5-dye fluorescence. Therefore, no IC50 or EC50 values could be determined for this conjugate. |
| Affinity data for this assay | |
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