KC-6272

Ba/F3-AXL Cell Line

61471

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Background of Ba/F3-AXL Cell Line

AXL is a transmembrane receptor tyrosine kinase, belonging to the TAM kinase family. AXL is expressed in bone marrow, myeloid cells, also found in tumor cells and tumor vasculature. The interaction with its ligand GAS6 can stimulate cell proliferation and survival. AXL has been implicated as a cancer diver gene and its expression level is correlated with the prognosis of several aggressive tumors.
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-6272
Cell Line Name	Ba/F3-AXL Cell Line
NCBI/UniProt Accession Number	NM_001699.3
Clone Number	21#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous AXL gene
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 +1μg/mL Puromycin+8ng/mL mIL-3
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

Cell Line Generation

Ba/F3-AXL Cell Line was generated using a lentiviral vector expressing AXL sequence.

Characterization

Figure 1: Characterization of AXL overexpression in the Ba/F3-AXL stable clone using FACS.

Figure 2: Characterization of AXL in the Ba/F3 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS +1μg/mL Puromycin+8ng/mL mIL-3)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% RPMI1640 + 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. Wilson, C, X Ye, T Pham, E Lin, S Chan, E McNamara, R M Neve, et al. 2014. AXL Inhibition Sensitizes Mesenchymal Cancer Cells to Antimitotic Drugs. Cancer Research 74 (20): 5878–90.
2. Brand, Toni M, Mari Iida, Kelsey L Corrigan, Cara M Braverman, John P Coan, Bailey G Flanigan, Andrew P Stein, et al. 2017. The Receptor Tyrosine Kinase AXL Mediates Nuclear Translocation of the Epidermal Growth Factor Receptor. Sci. Signal. 10 (460). American Association for the Advancement of Science: eaag1064.