KC-3935

NCI-H1975-EGFR-T790M-C797S-L858R Cell Line

40338

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Background of NCI-H1975-EGFR-T790M-C797S-L858R 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). The overexpression or overactivity of EGFR has 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 rapid development of anticancer therapeutics agents, including Gefitinib, Erlotinib, Afatinib, Osimertinib (AZD9291) and Cetuximab.

Specifications

Catalog Number	KC-3935
Cell Line Name	NCI-H1975-EGFR-T790M-C797S-L858R Cell Line
Host Cell Line	NCI-H1975
Description	NCI-H1975 cell line stably expressing exogenous EGFR T790M/C797S/L858R 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 1 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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 42 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

NCI-H1975 EGFR T790M/C797S/L858R overexpressed cell Line was generated through plasmid transfection with exogenous EGFR T790M/C797S/L858R Sequence.

Characterization

Figure 1: Characterization of EGFR T790M/C797S/L858R overexpression in the NCI-H1975 stable clone using WB.

Figure2: Characterization of NCI-H1975-EGFR-T790M-C797S-L858R cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 1 μg/mL puromycin) 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 9.0 mL 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 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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.Thress, K. S. et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in nonÿsmall cell lung cancer harboring EGFR T790M. Nature Medicine 21, 560ÿ562 (2015). 2.Jia, Y. et al. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors. Nature 534, 129ÿ132 (2016).