KC-0178

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

Home » NCI-H1975-EGFR-L858R-T790M-C797S 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). 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-0178
Cell Line Name:
NCI-H1975-EGFR-L858R-T790M-C797S Cell Line
Host Cell Line:
Human NCI-H1975 cell line
Description:
NCI-H1975 cell line bearing exogenous EGFR T790M/C797S/L858R mutations
Quantity:
One vial of frozen cells (5×106 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+2µg/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:
Mycoplasma Status:
Negative

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: Characterization of EGFR and its mutants through WB

Application

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 2µg/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 9.0mL 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 1:3-1:6 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. 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).
Please enable JavaScript in your browser to complete this form.