KC-5694

CHOK1-GPR75 Cell Line

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Background of CHOK1-GPR75 Cell Line

GPR75 is a member of the G protein-coupled receptor family. GPRs are cell surface receptors that activate guanine-nucleotide binding proteins upon the binding of a ligand.

Specifications

Catalog Number	KC-5694
Cell Line Name	CHOK1-GPR75 Cell Line
Clone Number	10#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous GPR75 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 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

CHOK1-GPR75 cell line was generated using a lentiviral vector expressing the GPR75 sequence.

Characterization

Figure 1: Characterization of GPR75 overexpressing in CHOK1-GPR75 stable clone using FACS.

Figure 2: Characterization of GPR75 overexpressing in CHOK1-GPR75 stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Dashti MR, Gorbanzadeh F, Jafari-Gharabaghlou D, Farhoudi Sefidan Jadid M, Zarghami N. G Protein-Coupled Receptor 75 (GPR75) As a Novel Molecule for Targeted Therapy of Cancer and Metabolic Syndrome. Asian Pac J Cancer Prev. 2023 May 1;24(5):1817-1825. doi: 10.31557/APJCP.2023.24.5.1817. PMID: 37247305; PMCID: PMC10495892.
Cárdenas S, Colombero C, Panelo L, Dakarapu R, Falck JR, Costas MA, Nowicki S. GPR75 receptor mediates 20-HETE-signaling and metastatic features of androgen-insensitive prostate cancer cells. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Feb;1865(2):158573. doi: 10.1016/j.bbalip.2019.158573. Epub 2019 Nov 21. PMID: 31760076; PMCID: PMC6957769.