KC-1245

Ba/F3-ETV6-FGFR4-N535D-Cell-Line

21783

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Background of Ba/F3-ETV6-FGFR4-N535D-Cell-Line

FGFR4 is a member of the fibroblast growth factor receptor family which play a role in mitogenesis and differentiation. FGFR4 preferentially binds acidic fibroblast growth factor and is overexpressed in gynecological tumor samples, suggesting a role in breast and ovarian tumorigenesis. FGFR4 gene expression is up-regulated in doxorubicin-treated, apoptosisresistant cancer cell clones. Ectopic expression of FGFR4 in cancer cells leads to reduced apoptosis sensitivity on treatment with doxorubicin or cyclophosphamide, whereas knockdown of endogenous FGFR4 expression in breast cancer cell lines has the opposite effect. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-1245
Cell Line Name	Ba/F3-ETV6-FGFR4-N535D-Cell-Line
Description	Stable Ba/F3 clone expressing exogenous ETV6-FGFR4 fusion protein bearing N535D mutation in FGFR4 part.
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+1µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3 ETV6-FGFR4-N535D cell Line was generated using retrovirus vector expressing human ETV6-FGFR4-N535D sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 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 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

French, Dorothy M, Benjamin C Lin, Manping Wang, Camellia Adams, Theresa Shek, Kathy H?tzel, Brad Bolon, et al. 2012. ¡ùTargeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models.¡ì Edited by Maria G Castro. PLoS ONE 7 (5): e36713Ã¿12.
Roidl, A. et al: Resistance to chemotherapy is associated with fibroblast growth factor receptor 4 upÛåregulation. Clin Cancer Res. 2009 Mar 15;15(6):2058Ûå66.