KC-4585

293T-TSHR Cell Line

60393

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Background of 293T-TSHR Cell Line

TSHR (Thyroid Stimulating Hormone Receptor) is a G protein-coupled receptor that plays a critical role in thyroid function by mediating the effects of thyroid-stimulating hormone (TSH). It is primarily expressed in thyroid follicular cells, where it regulates thyroid hormone production and release. Activation of TSHR stimulates iodine uptake, thyroglobulin synthesis, and overall thyroid growth. Mutations in the TSHR gene are associated with various thyroid disorders, including congenital hypothyroidism, Graves' disease, and non-autoimmune hyperthyroidism.

Specifications

Catalog Number	KC-4585
Cell Line Name	293T-TSHR Cell Line
NCBI/UniProt Accession Number	NM_000369.5
Clone Number	NA
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous TSHR 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-TSHR cell line was generated using lentivirus expressing TSHR sequence.

Characterization

Figure 1. Characterization of TSHR over-expression in the 293T-TSHR stable clone using FACS.(Primary antibody: M22-hIgG1, Cat#KB-1360, Kyinno)

Figure 2. Characterization of TSHR over-expression in the 293T-TSHR stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 1μg/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% 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

Smith TJ. TSHR as a therapeutic target in Graves' disease. Expert Opin Ther Targets. 2017 Apr;21(4):427-432. doi: 10.1080/14728222.2017.1288215. Epub 2017 Feb 6. PMID: 28127991; PMCID: PMC5718165.
Huth S, Jaeschke H, Schaarschmidt J, Paschke R. Controversial constitutive TSHR activity: patients, physiology, and in vitro characterization. Horm Metab Res. 2014 Jun;46(7):453-61. doi: 10.1055/s-0034-1375651. Epub 2014 May 20. PMID: 24845969.
Huth S, Jaeschke H, Schaarschmidt J, Paschke R. Controversial constitutive TSHR activity: patients, physiology, and in vitro characterization. Horm Metab Res. 2014 Jun;46(7):453-61. doi: 10.1055/s-0034-1375651. Epub 2014 May 20. PMID: 24845969.