KC-5453

293T-GPR75 cell line

56945

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Background of 293T-GPR75 cell line

GPR75 (G Protein-Coupled Receptor 75) is a Protein Coding gene. Among its related pathways are Class A/1 (Rhodopsin-like receptors) and 15q13.3 copy number variation syndrome. Gene Ontology (GO) annotations related to this gene include G protein-coupled receptor activity and C-C chemokine receptor activity. An important paralog of this gene is TACR3. 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-5453
Cell Line Name	293T-GPR75 cell line
Host Cell Line	293T
Description	Stable HEK293T clone 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/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

293T GPR75 cell line was generated using lentiviral vector expressing GPR75 sequence

Characterization

Figure 1: Characterization of GPR75 overexpression in the 293T GPR75 stable clone using FACS.

Figure 2: Characterization of GPR75 overexpression in the 293T-GPR75 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1ug/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 x 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 1:4 ~ 1:8 every 2~3 days; seed out at about 1-3 x 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 x g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3 x10⁶cells/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.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.
2.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.
3.Garcia V, Gilani A, Shkolnik B, Pandey V, Zhang FF, Dakarapu R, Gandham SK, Reddy NR, Graves JP, Gruzdev A, Zeldin DC, Capdevila JH, Falck JR, Schwartzman ML. 20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension. Circ Res. 2017 May 26;120(11):1776-1788. doi: 10.1161/CIRCRESAHA.116.310525. Epub 2017 Mar 21. PMID: 28325781; PMCID: PMC5446268.