Bruton's tyrosine kinase (abbreviated BTK) is a tyrosine kinase, which plays a key role in B cell development and maturation. The identification of BTK as driver gene has led to repaid development of anti cancer therapeutic agents, including ibrutinib, which approved for B cell cancers like mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström's macroglobulinemia in 2013.
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.
Cell Line Name:
Ba/F3 ETV6-BTK-C481S-F517L Cell Line
Host Cell Line:
Mouse Ba/F3 cell line
Stable Ba/F3 clone expressing exogenous ETV6-BTK fusion gene with C481S and F517L double mutations.
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% RPMI1640+20% FBS+10% DMSO
Mostly single, round (some polymorph) cells in suspension
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 20 hours
Cell Line Generation
Ba/F3 ETV6-BTK-C481S-F517L cell Line was generated using ibrutinib stepwise exposure method.
Characterization using PCR sequencing and WB
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.
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
1. Akinleye, Akintunde, Yamei Chen, Nikhil Mukhi, Yongping Song, and Delong Liu. 2013. “Ibrutinib and Novel BTK Inhibitors in Clinical Development.” Journal of Hematology & Oncology 6 (1). Journal of Hematology & Oncology: 1–1. doi:10.1186/1756-8722-6-59.
2. Cheng, S, A Guo, P Lu, J Ma, M Coleman, and Y L Wang. 2014. “Functional Characterization of BTKC481S Mutation That Confers Ibrutinib Resistance: Exploration of Alternative Kinase Inhibitors,” September. Nature Publishing Group, 1–25. doi:10.1038/leu.2014.263.
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