KC-2638

Ba/F3-ETV6-BTK-C481S-F517L-Cell-Line

24460

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Background of Ba/F3-ETV6-BTK-C481S-F517L-Cell-Line

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.

Specifications

Catalog Number	KC-2638
Cell Line Name	Ba/F3-ETV6-BTK-C481S-F517L-Cell-Line
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 clone expressing exogenous ETV6-BTK fusion gene with C481S/F517L double 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	Yes

Cell Line Generation

Ba/F3 ETV6-BTK-C481S-F517L cell Line was generated using ibrutinib stepwise exposure method.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10%FBS) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Akinleye, Akintunde, Yamei Chen, Nikhil Mukhi, Yongping Song, and Delong Liu. 2013. ¡ùIbrutinib and Novel BTK Inhibitors in Clinical Development.¡ì Journal of Hematology & Oncology 6 (1). Journal of Hematology & Oncology: 1Ã¿1. doi:10.1186/1756-8722-6-59.
Cheng, S, A Guo, P Lu, J Ma, M Coleman, and Y L Wang. 2014. ¡ùFunctional Characterization of BTKC481S Mutation That Confers Ibrutinib Resistance: Exploration of Alternative Kinase Inhibitors,¡ì September. Nature Publishing Group, 1Ã¿25. doi:10.1038/leu.2014.263.