KC-4486

NIH/3T3-PDGFRb-N666S-K976N-Cell-Line-Cell-Line

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Background of NIH/3T3-PDGFRb-N666S-K976N-Cell-Line-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-4486
Cell Line Name	NIH/3T3-PDGFRb-N666S-K976N-Cell-Line-Cell-Line
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 clone expressing exogenous PDGFRb gene bearing N666S-K976N 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

Cell Line Generation

NIH/3T3 PDGFRb-N666S-K976N cell line was generated using a lentiviral vector expressing the human PDGFRb-N666S-K976N sequence.

Characterization

Figure 1: Characterization of human PDGFRb-N666S-K976N 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 1:4-1:6 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

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.