KC-5258

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

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Background of NCI-H1975-EGFR-L858R-T790M-C797S-KI Cell Line

The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor, thus inducing receptor dimerization and tyrosine autophosphorylation leading to cell proliferation. Mutations in this gene are associated with lung cancer. EGFR is a component of the cytokine storm which contributes to a severe form of Coronavirus Disease 2019 (COVID-19) resulting from infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Specifications

Catalog Number	KC-5258
Cell Line Name	NCI-H1975-EGFR-L858R-T790M-C797S-KI Cell Line
Clone Number	1C1
Host Cell Line	NCI-H1975
Description	Stable NCI-H1975 clone expressing exogenous EGFR gene bearing L858R-T790M-C797S mutations, No.1C1
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	NA
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 30 hours
Mycoplasma Status	Negative

Cell Line Generation

NCI-H1975-EGFR-L858R-T790M-C797S-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of NCI-H1975-EGFR-L858R-T790M-C797S-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of NCI-H1975-EGFR-L858R-T790M-C797S-KI cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of dose-response curves for EGFR inhibitors on NCI-H1975 and NCI-H1975-EGFR-L858R-T790M-C797S-KI cells.

Figure 4: Characterization of NCI-H1975-EGFR-L858R-T790M-C797S-KI cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 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 1:3-1:6 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

Koletsa T, Kotoula V, Karayannopoulou G, Nenopoulou E, Karkavelas G, Papadimitriou CS, Kostopoulos I. EGFR expression and activation are common in HER2 positive and triple-negative breast tumours. Histol Histopathol. 2010 Sep;25(9):1171-9. doi: 10.14670/HH-25.1171. PMID: 20607659.
Xu L, Hausmann M, Dietmaier W, Kellermeier S, Pesch T, Stieber-Gunckel M, Lippert E, Klebl F, Rogler G. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines. BMC Cancer. 2010 Jun 18;10:302. doi: 10.1186/1471-2407-10-302. PMID: 20565817; PMCID: PMC2896958.