KC-1263

BaF3 ERBB2-V777L Cell Line

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Background of BaF3 ERBB2-V777L Cell Line

HER2, also named ERBB2, 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. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, 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.

Specifications

Catalog Number	KC-1263
Cell Line Name	BaF3 ERBB2-V777L Cell Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous ERBB2 gene bearing V777L mutation
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
Selection Marker	Puromycin
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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3 ERBB2-V777L cell Line was generated using retrovirus vector expressing ERBB2-V777L sequence.

Characterization

Figure 1: Characterization of ERBB2 mutation in the Ba/F3 stable clone using FACS.

Figure 2: Ba/F3 cells expressing ERBB2 mutant were seeded into 96-well plates, treated with compounds for 72 hours, and then read out with Cell-Titer Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 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 1:10 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. 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. 2.2.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.