KC-4853

CT26-PDL1-EGFR Cell Line

60615

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Background of CT26-PDL1-EGFR Cell Line

PD-L1, also called human programmed cell death ligand 1, is a transmembrane protein that plays a significant rolein suppressing the immune system during particular events such as pregnancy, tissue allografts, autoimmune disease, virus infection, and cancer. PD-L1 binds to its receptor PD-1 on activated T cells, B cells, and myeloid cells to modulate activation or inhibition. Upregulation of PD-L1 can allow the cancer cell to evade the host immune system.
EGFR (Epidermal Growth Factor Receptor) is a Protein Coding gene. 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-4853
Cell Line Name	CT26-PDL1-EGFR Cell Line
NCBI/UniProt Accession Number	NM_005228\|NM_014143
Clone Number	11-2#
Host Cell Line	CT26
Description	Stable CT26 clone expressing exogenous PDL1-EGFR 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 + 400μg/mL Hygromycin B
Selection Marker	Hygromycin B
Morphology	Fibroblastoid
Subculture	Split saturated culture 1:4-1:8 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

CT26-PDL1-EGFR cell line was generated using a lentiviral vector expressing the PDL1-EGFR sequence.

Characterization

Figure 1: Characterization of PDL1-EGFR overexpression in the CT26-PDL1-EGFR stable clone using FACS.(Primary antibody: Cetuximab, Cat#KB-1106, Kyinno)

Figure 2: Characterization of PDL1 overexpressing in CT26-PDL1-EGFR stable clone using PCR sequencing.

Figure 3: Characterization of EGFR overexpressing in CT26-PDL1-EGFR stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 400μg/mL Hygromycin B)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 1:4-1:8 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

Ning H, Chiu SH, Xu X, Ma Y, Chen JL, Yang G. The Immunosuppressive Roles of PD-L1 during Influenza A Virus Infection. Int J Mol Sci. 2023 May 11;24(10):8586.
Lei Q, Wang D, Sun K, Wang L, Zhang Y. Resistance Mechanisms of Anti-PD1/PDL1 Therapy in Solid Tumors. Front Cell Dev Biol. 2020 Jul 21;8:672.
Yang M, Xu X, Cai J, Ning J, Wery JP, Li QX. NSCLC harboring EGFR exon-20 insertions after the regulatory C-helix of kinase domain responds poorly to known EGFR inhibitors. Int J Cancer. 2016 Jul 1;139(1):171-6. doi: 10.1002/ijc.30047. Epub 2016 Mar 25. PMID: 26891175.