KC-4229

NIH/3T3-AXL-Low Cell Line

44141

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Background of NIH/3T3-AXL-Low 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.

Specifications

Catalog Number	KC-4229
Cell Line Name	NIH/3T3-AXL-Low Cell Line
NCBI/UniProt Accession Number	NM_021913.5
Clone Number	5-2#
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 cell line 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM +10%FBS+2µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 2-3 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

NIH/3T3-AXL-Low Cell Line was generated using a lentiviral vector expressing the AXL sequence.

Characterization

Figure 1: Characterization of AXL overexpression in NIH/3T3 stable clone using FACS.

Figure 2: Characterization of NIH/3T3-AXL-Low Cell Line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 2μg/mL Puromycin)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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

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.
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.