| Assay Method Information | |
| | Inhibition Assays of Transporters (hSERT and rVMAT2) |
| Description: | hSERT and rVMAT2 fluorometric screening assays. For both hSERT and rVMAT2 screening experiments, respective singly transfected cells were seeded at a density of 0.09×106 cells/well in poly-D-Lysine (Alamanda Polymers, Inc.) coated white solid-bottom 96-well plates (Costar). Growth was permitted for approximately 44 hours in said aqueous media and at an incubation environment of 37° C. and 5% Carbon Dioxide. At the beginning of the experiment, the cellular growth solution was aspirated, and individual cells were rinsed with 150 μL of 1×Dulbecco's Phosphate Buffered Saline (PBS; HyClone). 63 μL of Experimental Media (consisting of the following contents: DMEM without phenol red but with 4.5 g/L of D-Glucose (Gibco), 1% (v/v) FBS (Atlanta Biologicals), 100 U/mL Penicillin (Gibco), and 10 μg/mL Streptomycin (Gibco)) with 2×tiered concentrations of inhibitor (or DMSO, the vehicle of these experiments) were added to the respective wells. Control inhibitors used in these studies include Imipramine for hSERT experiments, and Reserpine for rVMAT2 experiments (Eiden, L. E. and Weihe, E. 2011; Sette, M. et al. 1983). At the conclusion of the pre-incubation period (60 minutes for hSERT experiments and 30 minutes for rVMAT2 experiments), 63 μL of Experimental Media containing 2×various concentrations of tested inhibitor (or vehicle) along with a specified amount of fluorescent substrate, APP+(Karpowicz, R. J. et al 2013) (final concentration: 1.1 μM for hSERT experiments) or FFN206 (Hu, G. et al. 2013) (final concentration: 0.75 μM for rVMAT2 experiments) were added to the present solution contained within the wells. After a required incubation period (30 minutes for hSERT experiments and 60 minutes for rVMAT2 experiments) for proper fluorescent probe uptake, the contents of each well were aspirated and consequently, rinsed twice with 120 μL of PBS. A final solution of 120 μL of PBS is finally added to all corresponding wells for cell maintenance before undergoing fluorescence uptake reading by a BioTek H1MF plate reader. The excitation and emission wavelengths of APP+ were set at 389 and 442 nm, respectively. Alternatively, the excitation and emission wavelengths of FFN206 were designed at 370 and 464 nm, respectively. |
| Affinity data for this assay | |
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