kc-5706

Ba/F3-EGFR-D770delinsEQPP Cell Line

58996

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Background of Ba/F3-EGFR-D770delinsEQPP Cell Line

EGFR (Epidermal Growth Factor Receptor) is a receptor tyrosine kinase that is a critical driver of cell proliferation and survival. Its aberrant activation, through overexpression or mutations, is a well-established oncogenic event in multiple cancers, particularly non-small cell lung cancer (NSCLC). This has made EGFR a premier drug target, leading to the development of tyrosine kinase inhibitors (TKIs). A central focus of current EGFR research is understanding and overcoming the inevitable drug resistance that arises to these targeted therapies.

Specifications

Catalog Number	kc-5706
Cell Line Name	Ba/F3-EGFR-D770delinsEQPP Cell Line
Clone Number	2#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 cell line expressing exogenous EGFR-D770delinsEQPP gene
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% RMPI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640+10%FBS
Selection Marker	NA
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:4-1:10 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 20 hours
Mycoplasma Status	Negative

Cell Line Generation

Ba/F3-EGFR-D770delinsEQPP Cell Line was generated using a retrovirus vector expressing the EGFR-D770delinsEQPP sequence.

Characterization

Figure 1: Characterization of EGFR-D770delinsEQPP overexpression in the Ba/F3-EGFR-D770delinsEQPP stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (RPMI1640 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 1:4-1:10 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Arrieta O, Cardona AF, Corrales L, Campos-Parra AD, Sánchez-Reyes R, Amieva-Rivera E, Rodríguez J, Vargas C, Carranza H, Otero J, Karachaliou N, Astudillo H, Rosell R; CLICaP. The impact of common and rare EGFR mutations in response to EGFR tyrosine kinase inhibitors and platinum-based chemotherapy in patients with non-small cell lung cancer. Lung Cancer. 2015 Feb;87(2):169-75. doi: 10.1016/j.lungcan.2014.12.009. Epub 2014 Dec 18. PMID: 25558790.
Wheeler SE, Egloff AM, Wang L, James CD, Hammerman PS, Grandis JR. Challenges in EGFRvIII detection in head and neck squamous cell carcinoma. PLoS One. 2015 Feb 6;10(2):e0117781. doi: 10.1371/journal.pone.0117781. PMID: 25658924; PMCID: PMC4320077.