KC-3840

CT26-TSHR-Cell-Line

34882

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Background of CT26-TSHR-Cell-Line

TSHR, also known as LGR3, the protein encoded by this gene is a membrane protein and a major controller of thyroid cell metabolism. The encoded protein is a receptor for thyrothropin and thyrostimulin, and its activity is mediated by adenylate cyclase. Defects in this gene are a cause of several types of hyperthyroidism. Three transcript variants encoding different isoforms have been found for this gene. Mutations in the TSHR gene are mostly associated with thyroid dysgenesis, and prevent or disrupt normal development of the gland. Subclinical hyperthyroidism is defined biochemically as a low or undetectable thyroid-stimulating hormone (TSH) with normal thyroid hormone levels. Low TSHR signaling is considered to associate with cognitive impairment.

Specifications

Catalog Number	KC-3840
Cell Line Name	CT26-TSHR-Cell-Line
NCBI/UniProt Accession Number	NM_000369.5
Clone Number	5#
Host Cell Line	CT26
Description	Stable CT26 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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
In Vivo Validation	NA

Cell Line Generation

CT26 TSHR cell line was generated using a lentiviral vector expressing the TSHR sequence.

Characterization

Figure 1: Characterization of TSHR overexpression in the CT26 human TSHR stable clone using FACS.

Figure 2: Characterization of endogenous TSHR expression in CT26 cell using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Association of TSHR gene single nucleotide intronic polymorphism with the risk of hypothyroid and hyperthyroid disorders in Yazd province. Sci Rep. 2022 Sep 21;12(1):15745. 2. Molecular investigation of TSHR gene in Bangladeshi congenital hypothyroid patients. PLoS One. 2023 Aug 10;18(8):e0282553. 3. TSHR signaling promotes hippocampal dependent memory formation through modulating Wnt5a/β-catenin mediated neurogenesis. Biochem Biophys Res Commun. 2024 Apr 16;704:149723.