| Assay Method Information | |
| | Radioligand Competitive Binding Assay |
| Description: | The binding affinities of test compounds were determined by a radioligand competitive binding assay using 177Lu labeled C-1 as the reference radioligand.Briefly, 100 uL HT-29 cells expressing human NTSR-1 at a density of 1 2 106 cells/mL were mixed with binding buffer (RPMI-1640 medium supplemented with 0.25% bovine serum albumin) in each well of the 96-well filter plate (Millipore). Then the cells in each well were incubated with 177Lu C-1 (0.55 uCi/well) and the test compound under 37 C. for 1 hr (n=3). After incubation, unbound 177Lu C-1 and the test compound in each well was removed by filtration using a Multiscreen vacuum manifold (Millipore) and the cells were further rinsed with the binding buffer for 5 times. The cells from each well were then collected and radioactivity of the cells in each well was individually measured by y counter (2480 WIZARD2, PerkinElmer). The best-fit IC50 value of test compounds (inhibitory concentration when 50% of the bound 177Lu C-1 on cells were displaced) were calculated by fitting the data with nonlinear regression using GraphPad Prism 8.0.1. |
| Affinity data for this assay | |
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