KC-5403

NIH/3T3-PDGFRb-N666S-Cell-Line

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Background of NIH/3T3-PDGFRb-N666S-Cell-Line

The protein encoded by PDGFRb is a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. These growth factors are mitogens for cells of mesenchymal origin. The identity of the growth factor bound to a receptor monomer determines whether the functional receptor is a homodimer (PDGFB or PDGFD) or a heterodimer (PDGFA and PDGFB). PDGFRb is essential for normal development of the cardiovascular system and aids in rearrangement of the actin cytoskeleton. PDGFRb is flanked on chromosome 5 by the genes for granulocyte-macrophage colony-stimulating factor and macrophage-colony stimulating factor receptor; all three genes may be implicated in the 5-q syndrome. A translocation between chromosomes 5 and 12, that fuses this gene to that of the ETV6 gene, results in chronic myeloproliferative disorder with eosinophilia.

Specifications

Catalog Number	KC-5403
Cell Line Name	NIH/3T3-PDGFRb-N666S-Cell-Line
Clone Number	3-12#
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 clone expressing exogenous PDGFRB gene bearing N666S 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	DMEM0 + 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

Cell Line Generation

NIH/3T3-PDGFRb-N666S-cell-line was generated using a lentiviral vector expressing the PDGFRb-N666S sequence.

Characterization

Figure 1: Characterization of dose-response curves for PDGFRb inhibitors on NIH/3T3-PDGFRb-N666S cells.

Figure 2: Characterization of PDGFRb-N666S in the NIH/3T3-PDGFRb-N666S stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 2μg/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 1:4-1:6 every 2-3 days; seed out at about 1-3 × 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×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. Liu B, Xiao X, Lin Z, Lou Y, Zhao L. PDGFRB is a potential prognostic biomarker and correlated with immune infiltrates in gastric cancer. Cancer Biomark. 2022;34(2):251-264. doi: 10.3233/CBM-210335. PMID: 34958001.
2.Agaimy A, Bieg M, Michal M, Geddert H, Märkl B, Seitz J, Moskalev EA, Schlesner M, Metzler M, Hartmann A, Wiemann S, Michal M, Mentzel T, Haller F. Recurrent Somatic PDGFRB Mutations in Sporadic Infantile/Solitary Adult Myofibromas But Not in Angioleiomyomas and Myopericytomas. Am J Surg Pathol. 2017 Feb;41(2):195-203.