KC-5947

CT26-EGFR-GFP-Luc2-Low-Cell-Line

60678

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Background of CT26-EGFR-GFP-Luc2-Low-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), EGFR overexpression or overactivity have 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 development of anticancer therapeutics agents, including gefitinib, Erlotinib, Afatinib, Osimertinib (AZD9291) and cetuximab.
Luciferase is an oxidative enzyme that can produce bioluminescence with addition of luciferin, but doesn’t need an external light source unlike fluorescent proteins. Photo emission can be detected directly by light sensitive devices. Such as luminometers or modified microscopes. Luciferase is widely used in many fields of biological research, such as transcriptional activity, kinase or other enzyme activity, cellular ATP level, and whole animal imaging.
Green fluorescent protein (GFP) is a β - barrel protein 1 composed of 238 amino acids with a molecular weight of approximately 27 kDa. GFP was isolated from the crystal jellyfish Aequorea Victoria. GFP can convert the blue fluorescence emitted by jellyfish luminescent protein through chemical reactions into green fluorescence through energy transfer. The excitation wavelength of GFP is 488 nm, and there is an emission peak at approximately 507 nm.

Specifications

Catalog Number	KC-5947
Cell Line Name	CT26-EGFR-GFP-Luc2-Low-Cell-Line
NCBI/UniProt Accession Number	NM_005228
Clone Number	1#
Host Cell Line	CT26
Description	Stable CT26 clone expressing exogenous EGFR and GFP and Luciferase gene in low level.
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% RPMI 1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI 1640 + 10% FBS
Selection Marker	NA
Morphology	Fibroblast
Subculture	Split saturated culture 1:6-1:8 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 24 hours
Mycoplasma Status	Negative

Cell Line Generation

CT26-EGFR-GFP-Luc2-Low-cell-line was generated using a lentiviral vector expressing the EGFR-GFP-Luc2 sequence.

Characterization

Figure 1: Characterization of EGFR overexpression in the CT26-EGFR-GFP-Luc2-Low stable clone using FACS.

Figure 2: Characterization of the CT26-EGFR-GFP-Luc2-cell-line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure 3:Characterization of GFP overexpression in the CT26-EGFR-GFP-Luc2 stable clone using FACS.

Figure 4: Characterization of EGFR in the CT26-EGFR-GFP-Luc2-Low stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI 1640 supplemented with 10% FBS)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 1:6-1:8 every 2-3 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. Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352, 786ÿ792 (2005)
2.Greer LF, Szalay AA (2002). "Imaging of light emission from the expression of luciferases in living cells and organisms: a review". Luminescence. 17 (1): 43–74. doi:10.1002/bio.676. PMID 11816060.
3.Pieribone V, Gruber D. Aglow in the Dark: The Revolutionary Science of Biofluorescence. Cambridge: Belknap Press. 2006. ISBN 0-674-01921-0. OCLC 60321612. Popular science book describing history and discovery of GFP