KC-5572

EMT6-ERBB2-GFP-Luc2 Cell Line

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Background of EMT6-ERBB2-GFP-Luc2 Cell Line

ERBB2, also named HER2, is a cell-surface receptor tyrosine kinase, ERBB2 overexpression or overactivity have associated with a number of cancers, especially the breast cancer. The identification of ERBB2 as a driver gene has led to the development of anticancer therapeutics agents, including lapatinib, Neratinib and Herceptin.
Green fluorescence protein(GFP) is a protein composed with 238 amino acids and isolated from Aequorea victoria. GFP emits green fluorescence spontaneously. Luciferase is an oxidative enzyme that can produce bioluminescence with the addition of luciferin, but don’t need an external light source, which is different from fluorescent proteins. The bioluminescence can be detected directly by light sensitive device, such as luminometer or modified microscope. Luciferase is widely used in many fields of biological research, such as transcriptional activity, kinase or other enzyme activity, cellular ATP level, whole animal imaging.

Specifications

Catalog Number	KC-5572
Cell Line Name	EMT6-ERBB2-GFP-Luc2 Cell Line
NCBI/UniProt Accession Number	NM_004448
Clone Number	9#
Host Cell Line	EMT6
Description	Stable EMT6 clone expressing exogenous ERBB2 and luciferase and EGFP 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS
Selection Marker	NA
Morphology	Epithelial
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

EMT6-ERBB2-GFP-Luc2 cell line was generated using a lentiviral vector expressing the ERBB2 and luciferase and EGFP gene sequence.

Characterization

Figure 1: Characterization of ERBB2 overexpression EMT6 stable clone using FACS(Kyinno, Cat#KP-2007).

Figure 2: Characterization of GFP overexpression in the EMT6-ERBB2-GFP-Luc2 stable clone using FACS.

Figure 3: Characterization of the EMT6-ERBB2-GFP-Luc2 cell line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure 4: Characterization of EMT6-ERBB2-GFP-Luc2 cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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. Wang, Shizhen Emily, Archana Narasanna, Marianela Perez-Torres, Bin Xiang, Frederick Y Wu, Seungchan Yang, Graham Carpenter, Adi F Gazdar, Senthil K Muthuswamy, and Carlos L Arteaga. 2006. HER2 Kinase Domain Mutation Results in Constitutive Phosphorylation and Activation of HER2 and EGFR and Resistance to EGFR Tyrosine Kinase Inhibitors. Cancer Cell 10 (1). Elsevier: 25–38.
2. Robichaux, Jacqulyne P, Yasir Y Elamin, Zhi Tan, Brett W Carter, Shuxing Zhang, Shengwu Liu, Shuai Li, et al. 2018. Mechanisms and Clinical Activity of an EGFR and HER2 Exon 20–Selective Kinase Inhibitor in Non–Small Cell Lung Cancer. Nature Medicine, April. Springer US, 1–15.
3. De Santis R. Anti-ErbB2 immunotherapeutics: struggling to make better antibodies for cancer therapy. MAbs. 2020 Jan-Dec;12(1):1725346. doi: 10.1080/19420862.2020.1725346. PMID: 32054397; PMCID: PMC7039626.
4. Strickler JH, Yoshino T, Graham RP, Siena S, Bekaii-Saab T. Diagnosis and Treatment of ERBB2-Positive Metastatic Colorectal Cancer: A Review. JAMA Oncol. 2022 May 1;8(5):760-769. doi: 10.1001/jamaoncol.2021.8196. PMID: 35238866.