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Screen Quest™ Live Cell Dopamine receptor D1R cAMP Assay Service Pack
Product key features
- Real-time monitoring: Enables precise, live-cell measurement of intracellular cAMP changes in response to D1R activation.
- Convenient: Provides all necessary components for precise measurement of D1R-mediated cAMP changes.
- High-Throughput: Optimized for use with FLIPR, FDSS, and other fluorescence microplate readers, making it ideal for large-scale screening of D1R-targeting compounds.
- Simple workflow: Utilizes wash-free calcium sensitive dyes reducing assay complexity and time.
Product description
Dopamine receptor D1 (D1R) is a key member of the G-protein coupled receptor (GPCR) family, involved in several physiological processes, including motor control, reward, learning, and memory. Dysregulation of D1R signaling is implicated in various neurological disorders, such as Parkinson’s disease, schizophrenia, and drug addiction. Traditional methods for studying GPCRs may not fully capture the broad spectrum of signaling mechanisms that occur through specific receptors like D1R, which couple to adenylyl cyclase activity and intracellular cAMP production.
The Screen Quest™ Live Cell Dopamine Receptor D1 cAMP Assay Service Pack is designed for real-time, high-throughput monitoring of intracellular cAMP changes associated with D1R activation using transfected cell lines and Calbryte™ 520 wash-free calcium fluorescence detection methods. Unlike conventional assays that require cell lysis, this assay preserves cellular integrity, enabling both temporal and spatial resolution of specific signaling events associated with D1 receptors. This assay employs cell lines transfected to express D1R along with a promiscuous G-protein Gα16. The Gα16 protein allows D1R, which primarily signals through the cAMP pathway, to also couple to Gq signal transduction and mobilize intracellular calcium. Activation of D1R by specific ligands, such as dopamine, can be detected using calcium-sensitive dyes such as Calbryte™ 520 AM, Cal-520™ AM, Fluo-8™ AM, Fluo-4™ AM, or corresponding no-wash calcium kits. The inclusion of D1R and Gα16 co-expression ensures robust calcium signaling for reliable assay performance.
This service pack provides all necessary components for precise measurement of D1R-mediated cAMP changes using FLIPR, FDSS, or equivalent fluorescence microplate readers. It is an ideal tool for studying D1R signaling pathways and evaluating potential therapeutic compounds targeting this receptor, particularly in the context of neurological diseases, addiction research, and CNS-related disorders.
Example protocol
AT A GLANCE
Prepare cells for transfection
Prepare Transfectamine™ 5000-DNA mixture
Add Transfectamine™ 5000-DNA mixture to the cell culture, incubate overnight
Transfer the transfected cells to a 96-well plate 24-30 hours after transfection, and incubate the culture overnight
Add Calbryte™ 520 NW dye-loading solution
Incubate at room temperature or 37 °C for 30-60 minutes
Monitor the fluorescence intensity at Ex/Em = 490/525 nm
Thaw the kit components at room temperature before starting the experiment.
CELL PREPARATION
Seed the cells such that they will be ~60-70% confluent at the time of transfection.
Replace with fresh growth medium before transfection. For example, replace with 2 mL of medium per well for 6-well plates and 6 mL of medium for 10 cm plates.
PREPARATION OF STOCK SOLUTIONS
Add 20 µL of DMSO (Component F) into the vial of Calbryte™ 520NW (Component C), and mix them well.
Note: 20 µL of Calbryte™ 520NW stock solution is enough for one plate. Unused Calbryte™ 520NW stock solution can be aliquoted and stored at < -20 °C for more than one month, provided the tubes are tightly sealed. Protect from light and avoid repeated freeze-thaw cycles.
PREPARATION OF WORKING SOLUTION
- Combine 9 mL of HHBS (Component E) with 1 mL of 10X Pluronic® F127 Plus (Component D), and mix thoroughly.
Add 20 µL of Calbryte™ 520 NW stock solution to 10 mL of 1X Assay Buffer, and mix well.
Note: The working solution is stable for at least 2 hours at room temperature.
Add 15 µL of ddH2O to the vial of Ga16 DNA (Component A) and D1R DNA (Component G), to get the final concentration of 1 µg/µL for both DNAs.
Mix 3 µg of DNA [for example, 1.5 µg of Ga16 DNA (Component A) and 1.5 µg of D1R DNA (Component G)] with 200 µL of serum-free medium.
Add 9 µL of Transfectamine™ 5000 (Component B) to the mixture from Step 2.
Mix well and incubate at room temperature for 20 minutes.
Note: The ratio of Transfectamine™ 5000 and DNA need to be optimized for different cell lines. In general, the ratio for Transfectamine™ 5000 Transfection Reagent (µL) to DNA (µg) should be 3-5 µL : 1 µg.
Table 1. Sample protocols for a 6-well plate and a 10 cm plate
| Component | 6 well plate (per well) | 10 cm plate |
| Fresh culture medium | 2 mL | 6 mL |
| Plasmid | ~3 µg | 10~15 µg |
| Serum-free medium | 200 µL | 600 µL |
| Transfectamine™ 5000 Transfection Reagent | ~9 µL | ~30-45 µL |
SAMPLE EXPERIMENTAL PROTOCOL
Add Transfectamine™ 5000 -DNA mixture to the culture plate and incubate overnight.
Note: The recombinant protein can start to be detected as early as 16 hours after transfection. The maximal expression level may be observed 72~96 hours after transfection.
Transfer the transfected cells to a 96-well plate 24-30 hours post transfection and incubate overnight.
- For adherent cells: Plate cells overnight in the growth medium at 40,000 to 80,000 cells/well/100 µL for a 96-well plate.
- For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in cell growth medium or HHBS at 125,000 to 250,000 cells/well/100 µL for a 96-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments.
Note: Each cell line should be evaluated on the individual basis to determine the optimal cell density for the intracellular calcium mobilization.
Add 100 µL/well (96-well plate) of Calbryte™ 520NW working solution into the cell plate.
Incubate the dye-loaded plate in a cell incubator for 30 minutes, and then incubate the plate at room temperature for another 15-30 minutes.
Note: If the assay requires 37 °C, perform the experiment immediately without further room temperature incubation.
Note: If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1 hour (It is recommended that the incubation time be no longer than 2 hours.)
Prepare the compound plate with HHBS or your desired buffer.
Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 490/525 nm.
References
Authors: MacIsaac, Angela R and Wellington, Andrea J and Filicetti, Kyle and Eggers, Erika D
Journal: Experimental eye research (2024): 110049
Authors: Chen, Xiaowei and Liu, Xiaofeng and Luan, Shuxin and Wang, Xuxin and Zhang, Ying and Hao, Yulei and Zhang, Qiang and Zhang, Jiaming and Zhao, Hua
Journal: European archives of psychiatry and clinical neuroscience (2024): 867-878
Authors: Li, Yaqian and Wang, Qianwen and Zhang, Xueping and Zheng, Ruobing and Li, Derong and Wang, Yong
Journal: Brain research (2024): 149174
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Journal: Environmental toxicology and pharmacology (2024): 104417
Authors: Li, Jiajia and Pan, Chi and Huang, Bingwu and Qiu, Jiani and Jiang, Chenchen and Dong, Zhanglei and Li, Jun and Lian, Qingquan and Wu, Binbin
Journal: Addiction biology (2024): e13401
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