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	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+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).