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.
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
Cell Line Generation
Ba/F3 ERBB2-T798I-Q799E cell Line was generated using a retrovirus vector expressing the human ERBB2-T798I Q799E sequence.
Characterization using PCR sequencing and WB
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Application
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Cell Resuscitation
1. Prewarm culture medium (RPMI1640 + 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. 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.
