KC-2277

HEK293T-cAMP-biosensor-TAAR1-Cell-Line

23740

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Background of HEK293T-cAMP-biosensor-TAAR1-Cell-Line

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-2277
Cell Line Name	HEK293T-cAMP-biosensor-TAAR1-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	HEK293-cAMP-biosensor-22F cell line stable expressing exogenous TAAR1 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+100µg/mL Hygromycin B+9µg/mL Puromycin
Selection Marker	Puromycin, HygromycinB
Morphology	Epithelial
Subculture	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
In Vivo Validation	NA

Cell Line Generation

HEK293-cAMP-biosensor-hTAAR1 cell line was generated using lentivirus methods.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM+10%FBS + 100µg/mL Hygromycin B + 9µg/mL Puromycin) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

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.
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.