KC-2934

NIH/3T3-FGFR3-S249C-Cell-Line-Cell-Line

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Background of NIH/3T3-FGFR3-S249C-Cell-Line-Cell-Line

FGFR3 encodes a member of the fibroblast growth factor receptor (FGFR) family, with its amino acid sequence being highly conserved between members and among divergent species. FGFR family members differ from one another in their ligand affinities and tissue distribution. A full-length representative protein would consist of an extracellular region, composed of three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment and a cytoplasmic tyrosine kinase domain. The extracellular portion of the protein interacts with fibroblast growth factors, setting in motion a cascade of downstream signals, ultimately influencing mitogenesis and differentiation. This particular family member binds acidic and basic fibroblast growth hormone and plays a role in bone development and maintenance. Mutations in this gene lead to craniosynostosis and multiple types of skeletal dysplasia.

Specifications

Catalog Number	KC-2934
Cell Line Name	NIH/3T3-FGFR3-S249C-Cell-Line-Cell-Line
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 clone expressing exogenous FGFR3 gene bearing S249C mutation.
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 + 2μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblast
Subculture	Split saturated culture 1:4-1:6 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

NIH/3T3 FGFR3-S249C cell line was generated using a lentiviral vector expressing the human FGFR3-S249C sequence.

Characterization

Figure 1: Characterization of FGFR3 and its mutants overexpressing in NIH/3T3 stable clones using Western Blot.

Figure 2: Characterization of human FGFR3-S249C in the NIH/3T3 stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 2μg/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

Ascione CM, Napolitano F, Esposito D, Servetto A, Belli S, Santaniello A, Scagliarini S, Crocetto F, Bianco R, Formisano L. Role of FGFR3 in bladder cancer: Treatment landscape and future challenges. Cancer Treat Rev. 2023 Apr;115:102530. doi: 10.1016/j.ctrv.2023.102530. Epub 2023 Feb 28. PMID: 36898352.
Jing W, Wang G, Cui Z, Xiong G, Jiang X, Li Y, Li W, Han B, Chen S, Shi B. FGFR3 Destabilizes PD-L1 via NEDD4 to Control T-cell-Mediated Bladder Cancer Immune Surveillance. Cancer Res. 2022 Jan 1;82(1):114-129. doi: 10.1158/0008-5472.CAN-21-2362. Epub 2021 Nov 9. PMID: 34753771.