| Assay Method Information | |
| | Measurement of Human α7 Nicotinic Acetylcholine Receptor (nAChR)'s Activity |
| Description: | Activity of heteromeric α7 nAChR was measured via FlexStation-Ca2+ influx assay. In the present example, in consideration of α7 nAChR being Ca2+-permeable non-selective cationic channels, changes intracellular Ca2+ concentration were measured using a fluorescent dye Calcium-3 (available from Molecular Devices) and FlexStation II instrument (available from Molecular Devices).Human CHRNA7 (NM_000746) cDNA ORF clone (C/N RC221382; Origene) and Human RIC3 (NM_024557) cDNA ORF clone (C/N RC205179; Origene) were subcloned into pcDNA2.1/Zeo(+) vector (available from Invitrogen, Co.) to construct HEK293T/17 cells (ATCC, CRL-11268) transfected with human α7 nAChR. Afterward, the cells were suspended in growth media (consisted of Dulbecco's Modified Eagle's Media (DMEM, available from Invitrogen), a 10% heat-inactivated fetal bovine serum (FBS, available from Invitrogen), 300 μg/ml Geneticin (available from Invitrogen), 250 μg/ml Zeocin (available from Invitrogen), and 1× penicillin/streptomycin (available from Invitrogen)), followed by plating onto a Φ150 mm plate. Twenty-four hours prior to the start of the assay, grown cells in the suspension were collected, followed by centrifugation and further suspension at a concentration of 5×105 cells/mL in growth media. This cell suspension was dispensed to each well of a 96-well black plate (5×104 cells/well) with a poly-D-lysine-coated transparent bottom (available from Biocoat, BD). The plate with the cells were incubated at about 37° C. in 5% CO2 for about 24 hours.On the day of the assay, after removal of the growth media, the cells were washed once with an assay buffer (7 mM Tris-Cl, 20 mM HEPES, 20 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 4 mM CaCl2, 120 mM NMDG, 5 mM D-glucose, pH 7.4), followed by addition of about 100 ul per well of a Calcium-3 dye diluted with the assay buffer, and storage at room temperature for about 1 hour. A test compound (10 mM stock in 100% dimethyl sulfoxide (DMSO)) was diluted with the assay buffer to various concentrations, from the highest at about 40 μM to be lower by ⅓, and PNU-120596 (available from Sigma) for amplifying Ca2+ permeability signaling was diluted to about 30 μM with the assay buffer. Epibatidine (available from Sigma) in a final concentration of about 1 μM was used as a positive control group.To measure changes in intracellular Ca2+ concentration, after the plate was stored at room temperature for about 1 hour and the test compound dilution plate were put into FlexStation II equipment, fluorescence of the cells were measured for about 30 seconds prior to addition of drugs (the compounds), followed by addition of PNU-120596 and measurement of changes in fluorescence for about 120 seconds. After the cells were exposed to the test compound, changes in fluorescence for about 90 seconds were measured (excitation at 485 nm/emission at 525 nm). The largest fluorescence value at each concentration was recorded, and an EC50 of the test compound was determined using non-linear regression analysis with relative fluorescence values relative to the positive control group. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |