KC-4403

BaF3-EGFR-WT-TROP2 Cell Line

53490

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Background of BaF3-EGFR-WT-TROP2 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. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their nhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.TROP2 aliases is TACSTD2. TACSTD2 (Tumor Associated Calcium Signal Transducer 2) is a Protein Coding gene. This intronless gene encodes a carcinoma-associated antigen. This antigen is a cell surface receptor that transduces calcium signals. Mutations of this gene have been associated with gelatinous drop-like corneal dystrophy.

Specifications

Catalog Number	KC-4403
Cell Line Name	BaF3-EGFR-WT-TROP2 Cell Line
Host Cell Line	Ba/F3-EGFR-WT
Description	Ba/F3-EGFR-WT cell line stably expressing exogenous TROP2 gene
Quantity	One vial 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+1000 µg/mL G418+80 ng/mL EGF
Selection Marker	G418
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 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	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

BaF3-EGFR-WT-TROP2 cell Line was generated using retrovirus vector expressing EGFR-WT and TROP2 sequence.

Characterization

Figure 1: Characterization of TROP2 overexpression in the BaF3-EGFR-WT stable clone using FACS.

Figure2: Characterization of BaF3-EGFR-WT-TROP2 cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640+10%FBS+1000 µg/mL G418+80 ng/mL EGF) 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 1:10 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. Vidmar T, Pavšič M, Lenarčič B. Biochemical and preliminary X-ray characterization of the tumor-associated calcium signal transducer 2 (Trop2) ectodomain. Protein Expr Purif. 2013 Sep;91(1):69-76. doi: 10.1016/j.pep.2013.07.006. Epub 2013 Jul 17. PMID: 23872121.
2.Zhao P, Yu HZ, Cai JH. Clinical investigation of TROP-2 as an independent biomarker and potential therapeutic target in colon cancer. Mol Med Rep. 2015 Sep;12(3):4364-4369. doi: 10.3892/mmr.2015.3900. Epub 2015 Jun 8. PMID: 26059528.
3.Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352, 786–792 (2005).

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.