Cyclic adenosine monophosphate (cAMP) is a second messenger that mediates numerous biological responses. Intracellular cAMP levels are increased by activation of G(s)-coupled G protein-coupled receptors (GPCRs) and decreased by activation of G(i)-coupled GPCRs via the adenylyl cyclase. Studies on the cAMP dynamics provided useful scientific insights for drug development and treatment of cAMP-related diseases such as some cancers and prefrontal cortex disorders.
Specifications
Catalog Number:
KC-2470
Cell Line Name:
HEK293T cAMP biosensor membrane TAAR1 Cell Line
Host Cell Line:
Human HEK293T cell line
Description:
HEK293-cAMP-biosensor-22F cell line stable expressing exogenous TAAR1 gene with membrane signal peptide and HA tag
Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL
Incubation:
37 °C with 5% CO2
Storage:
Liquid nitrogen immediately upon receiving
Doubling Time:
Approximately 30 hours
Mycoplasma Status:
Negative
Cell Line Generation
HEK293-cAMP-biosensor-membrane-TAAR1 cell line was generated using a lentiviral vector expressing human TAAR1 sequence with membrane signal peptide and HA tag.
Characterization
Application
Cell Resuscitation
1. Prewarm culture medium (DMEM + 10%FBS + 100μg/mL Hygromycin B + 9μg/mL Puromycin) in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL.
Cell Freezing
1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage
References
1. Jesper Mosolff Mathiesen , Line Vedel, Hans Br?uner-Osborne. cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors. Methods Enzymol. 2013;522:191-207.
2. Namdoo Kim, Seunghan Shin, and Se Won Bae. cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins. Cardiovascular Toxicology. Biosensors (Basel). 2021 Feb; 11(2): 39.
