KC-1474

PC9-EGFR-Del19/T790M/C797S-Cell-Line

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Background of PC9-EGFR-Del19/T790M/C797S-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-1474
Cell Line Name	PC9-EGFR-Del19/T790M/C797S-Cell-Line
Host Cell Line	PC9
Description	PC9 cell line bearing exogenous EGFR Del19/T790M/C797S mutations
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+0.5µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

PC9 EGFR Del19/T790M/C797S/L858R overexpressed cell Line was generated through plasmid transfection with exogenous EGFR Del19/T790M/C797S Sequence

Characterization

Figure 1: Characterization of EGFR-Del19/T790M/C797S in PC9 stable clone using Western Blot.

Figure 2: Characterization of EGFR-Del19/T790M/C797S in PC9 stable clones using PCR sequencing.

Figure 3: PC9 cells expressing EGFR mutants were seeded into 96-well plates, treated with compounds for 72 hours, and then read out with Cell-Titer Glo system.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 0.5μg/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 1:3-1:6 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

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).
Jia, Y. et al. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors. Nature 534, 129ÿ132 (2016).