| Assay Method Information | |
| | Ca2+ Fluorometry |
| Description: | α7 cells cells stably expressing human α7 nAchR were cultured in the medium detailed above, and were split twice a week. For the fluorometric measurements of cytosolic Ca2+ ion concentration ([Ca2+]i) cells were seeded in 96-well microplates at a density of 60000 cells/well and maintained overnight in a tissue culture incubator at 37° C. under an atmosphere of 95% air/5% CO2. The plating medium was identical with the culture medium. 50 μl of the growth medium was aspirated with a cell washer (BioTek Elx405UCVWS). Then 50 μl/well Calcium 5 kit diluted 2-fold in assay buffer was added manually using an 8-channel pipette. After an incubation period (20 minutes, 37° C.) 50 μl/well assay buffer containing vehicle (DMSO, 4% added) or reference α7 PAMs (4×of the final concentration) were added manually and the cells were incubated for an additional 10 minutes at 37° C. Baseline and agonist-evoked [Ca2+]i-changes were monitored with FlexStation II (Molecular Devices, Sunnyvale, Calif.), a plate reader fluorometer with integrated 8-channel fluid addition capability. Fluorescence measurements were carried out at 37° C. The dye was excited at 485 nm, emission was sampled at 525 nm at 1.4-s intervals. Baseline was recorded for 20 seconds followed by agonist stimulation. 50 μl 4× concentrated agonist solution was added to the cells using the pipettor of FlexStation II and fluorescence was monitored for an additional 40 seconds. Final DMSO concentration was 1% for all treatments. To achieve this, a series of DMSO stock solutions were prepared from all test compounds. These stocks were stored under 0° C. and were further diluted in assay buffer to obtain the desired final concentration immediately before the measurement. Agonist and PAM concentration-response studies were conducted in the presence of saturating concentrations of PAMs (mostly PNU-120596, 5 μM) and agonists (mostly PNU-282987, 1 μM), respectively. Results were expressed as ΔF/F values using SoftMax Pro software (Molecular Devices), where F was the resting fluorescence preceding agonist application and ΔF was the increase in fluorescence at a given time (ΔF=maximum fluorescence intensity values after stimulation minus average fluorescence intensity values before stimulation). In all experiments, all treatments were measured in multiple wells in parallel, and the mean ΔF/F values were used for analysis. |
| Affinity data for this assay | |
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