KC-0117

Ba/F3-EGFR-C797S-G719D-Cell-Line

19863

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Background of Ba/F3-EGFR-C797S-G719D-Cell-Line

EGFR, the Epidermal growth factor receptor, is a cell-surface receptor tyrosine kinase, and activated by binding of its specific ligand, such as epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), EGFR overexpression or overactivity have associated with a number of cancers, including the lung cancer and colon cancer. The identification of EGFR as a driver gene has led to the development of anticancer therapeutics agents, including gefitinib, Erlotinib, Afatinib, Osimertinib (AZD9291) and cetuximab. 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-0117
Cell Line Name	Ba/F3-EGFR-C797S-G719D-Cell-Line
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 clone expressing exogenous EGFR gene bearing C797S amino acid 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS
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 EGFR-C797S cell Line was generated using retrovirus vector expressing human EGFR sequence with C797S mutation.

Characterization

Figure: Characterization of EGFR and its mutants overexpressing in Ba/F3 stable clones using FACS.

Harvest and seed the Ba/F3 cells expressing EGFR mutant in 96-well plate (3000 cells/90μL medium).
Next day, add 10μL 10× serially diluted compound solution each well and incubate the plates for another 72 hours.
Add 100μL Cell Titer-Glo each well, mixed and readout using Envision.
Plot the dose-responsive curve and fit the IC₅₀ (the centration of 50% inhibition of DMSO vehicle treated clones) using GraphPad Prism software (Version 5).

Cell Resuscitation

Prewarm culture medium (RPMI-1640 supplemented with 10% FBS)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

Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352, 786ÿ792 (2005)