KC-2398

Ba/F3-EGFR-Del19-T790M-L718Q Cell Line

Home » Ba/F3-EGFR-Del19-T790M-L718Q Cell Line

Background

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-2398
Cell Line Name:
Ba/F3 EGFR-Del19-T790M-L718Q Cell Line
Host Cell Line:
Mouse Ba/F3 cell line
Description:
Stable Ba/F3 clone expressing exogenous EGFR gene bearing Del19, T790M and L718Q triple mutationss.
Quantity:
One vial of frozen cells (5X106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze 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

Cell Line Generation

Ba/F3-EGFR-Del19-T790M-L718Q cell Line was generated using retrovirus vector expressing human EGFR-Del19- T790M-L718Q sequence.

Characterization

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Application

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Cell Resuscitation

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

Cell Freezing

1. Prepare the freezing medium (70% RPMI-1640 + 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. Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352, 786–792 (2005)

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