KC-1857

Ba/F3-BCR-ABL-F317V-Cell-Line

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Background of Ba/F3-BCR-ABL-F317V-Cell-Line

BCR-ABL, breakpoint cluster region–Abelson murine leukemia viral oncogene homolog 1, is an abnormal protein mostly found in chronic myelogenous leukemia (CML) due to the fusion of abnormal configuration of DNA where the abl gene is fused to the BCR gene, forming the Philadelphia chromosome; BCR-ABL and its mutants can promote and maintain the malignant behavior of the cancer cells. The identification of BCR-ABL as a driver gene has led to the rapid development of anticancer therapeutics agents, including Imatinib, Dasatinib, Ponatinib and Nilotinib. 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-1857
Cell Line Name	Ba/F3-BCR-ABL-F317V-Cell-Line
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 clone expressing exogenous BCR-ABL gene bearing F317V point mutation in ABL part.
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 BCR-ABL-F317V cell Line was generated using a retrovirus vector expressing human BCR-ABL fusion sequence bearing F317V point mutation in the ABL part.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI-1640 supplemented with 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

Weisberg, E., Manley, P. W., Cowan-Jacob, S. W., Hochhaus, A. & Griffin, J. D. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nat Rev Cancer 7, 345ÿ356 (2007).
Akahane, D., Tauchi, T., Okabe, S., Nunoda, K. & Ohyashiki, K. Activity of a novel Aurora kinase inhibitor against the T315I mutant form of BCR-ABL: In vitro and in vivostudies. Cancer Science 99, 1251ÿ1257 (2008).