KC-4504

Ba/F3-EML4-ALK-R1275Q-Cell-Line

46460

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Background of Ba/F3-EML4-ALK-R1275Q-Cell-Line

EML4-ALK, echinoderm microtubule-associated protein-like 4 - anaplastic lymphoma kinase, is an abnormal protein with transforming activity found in some primary lung malignant tumors due to the fusion of abnormal configuration of DNA wherein the echinoderm microtubule-associated protein-like 4 (EML4) gene is fused to the anaplastic lymphoma kinase (ALK) gene; EML4-ALK and its mutants can promote and maintain the malignant behavior of the cancer cells. Identifying ALK as a driver gene has led to the rapid development of anticancer therapeutic agents, including crizotinib, ceritinib, and Brigatinib. 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-4504
Cell Line Name	Ba/F3-EML4-ALK-R1275Q-Cell-Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous EML4-ALK gene bearing R1275Q 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 EML4-ALK-R1275Q cell line was generated using retrovirus vector expressing human EML4-ALK-R1275Q sequence.

Characterization

Figure 1: Characterization of EML4-ALK mutation in the Ba/F3 stable clone using PCR sequencing.

Figure 2: Characterization of IC50 (Half Inhibitory Concentration) in Ba/F3-EML4-ALK-R1275Q stable clone using GraphPad Prism software.

Cell Resuscitation

Prewarm culture medium (RPMI1640 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

Koivunen, J. P. et al. EML4-ALK Fusion Gene and Efficacy of an ALK Kinase Inhibitor in Lung Cancer. Clinical Cancer Research 14, 4275ÿ4283 (2008).
Choi, Y. L. et al. Identification of Novel Isoforms of the EML4-ALK Transforming Gene in Non-Small Cell Lung Cancer. Cancer Research 68, 4971ÿ4976 (2008). 3. Gainor, J. F. et al. Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALKRearranged Lung Cancer. Cancer Discovery 1ÿ48 (2016). doi:10.1158/2159-8290.CD-16-059.