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.
CCR8, also named as CDw198, is a GPCR protein belonging to the beta chemokine receptor family, and is mainly expressed in the thymus. CCR8 play an important role in regulation of monocyte chemotaxis and thymic cell apoptosis after interaction with its ligand CCL1.
Specifications
Catalog Number:
KC-2317
Cell Line Name:
HEK293-cAMP-biosensor-CCR8-High Cell Line
Host Cell Line:
Human HEK293T cell line
Description:
HEK293-cAMP-biosensor-22F cell line stable expressing exogenous CCR8 gene
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-CCR8 cell line was generated using lentivirus methods.
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. Roos RS, Loetscher M, Legler DF, Clark-Lewis I, Baggiolini M, Moser B (1997). "Identification of CCR8, the receptor for the human CC chemokine I-309". J. Biol. Chem. 272 (28): 17251ÿ4.
2. 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.
3. 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.
