Neurotransmitter Transporter Uptake Assay Kit
Until recently, radioactively labeled compounds were used to measure serotonin, norepinephrine and dopamine transporter uptake. With the introduction of the Neurotransmitter Transporter Uptake Assay Kit, researchers now have a tool to screen for live-cell kinetic uptake for these three key neurotransmitters.
- Homogeneous, live-cell fluorescent assay replaces radioactive assays, reducing disposal costs and eliminating safety risks associated with radiolabeled assays
- One-step, homogeneous assay format is amenable to automation and high-throughput screening
- Two assay setup options offer greater flexibility: endpoint mode for high-throughput applications, or real-time kinetic mode for mechanistic studies
Data of Neurotransmitter Transporter Uptake Assay Kit
Nisoxetine inhibition in HEK-hSERT cells
HEK cells stably expressing human SERT were plated O/N at 10,000 cells per well in poly D-lysine-coated 384-well plates. Medium was removed and nisoxetine (published Ki = 383 nM) in HBSS/0.1% BSA was incubated with cells for 10 minutes at 37°C prior to dye addition. The assay was performed according to the Experimental Protocol and read on Molecular Devices' FlexStation® 3 Reader in kinetic mode for 30 minutes.
HEK cells stably expressing human NET were plated overnight (open blue circles) or on the day of assay (filled black squares) at 20,000 cells per well in poly D-lysine-coated 384-well plates and allowed to adhere for 3 hours. Medium was removed and nomifensine in HBSS/0.1% BSA was incubated with cells for 10 minutes at 37°C prior to dye addition. The assay was read on the FlexStation® 3 Reader in kinetic mode for 30 minutes. The inhibition curve is expressed as area under the curve.
IC50 correlation for DAT, NET and SERT (customer results)
Cells stably expressing human DAT, NET and SERT were plated overnight at 60.000 cells/well (96-well plate) and treated with 8 different known inhibitors. After 10 minutes the Dye Solution was added and the assay was run according the protocol described above. The log Ki was calculated from the resulting data using the Cheng-Prusoff equation and a correlation with known Ki values for all three transporters was determined. The correlation coefficients R2 for each transporter is presented in the graph, the correlation coefficient (R2 value) over all 24 values was 0.91.
Technology of Neurotransmitter Transporter Uptake Assay Kit
The Neurotransmitter Transporter Uptake Assay Kit uses a fluorescent substrate that mimics the biogenic amine neurotransmitters and is taken into the cell through those specific transporters, resulting in increased intracellular fluorescence intensity that is monitored in real time using a bottom-reading microplate reader. In this convenient no-wash procedure, cells are incubated with the kit reagent and transferred to the plate reader for evaluation. The assay is ideal for use in academic, biotech, and pharmaceutical environments for research, assay development, and screening.
For more details, please refer to the Neurotransmitter Transporter Uptake Assay Kit product insert, which may be accessed through the Molecular Devices Knowledge Base.
Resources of Neurotransmitter Transporter Uptake Assay Kit
Neurotransmitter transporters have become important pharmaceutical targets, as they play a key role in depression and neurodegenerative diseases (NDD) such as Alzheimer’s and Parkinson’…
Read this PDF to know how fluorescence-based assay kit is useful for the measurement of neurotransmitter transporter uptake activity in cell lines stably expressing the human DAT, NET,…
Here we present a homogeneous assay of norepinephrine, dopamine, and serotonin transporter activity based on cellular uptake of a fluorescent dye coupled with a proprietary masking dye.…
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