KC-0280

Ba/F3-FLT3-ITD-AXL-Cell-Line

20157

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Background of Ba/F3-FLT3-ITD-AXL-Cell-Line

Fms like tyrosine Kinase 3 (FLT3), also named as CD135, FLK2, is receptor kinase receptor, expressed on the surface of many hematopoietic progenitor cells. Overactivation of FLT3-ITD-AXL due to mutation and overexpression can lead to the development and poor prognosis of Leukemia, the identification of FLT3 as a driver gene has led to the repaid development of anti-AML therapeutics, including Sunitinib, Sorafenib and Quizartinib (AC220). 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-0280
Cell Line Name	Ba/F3-FLT3-ITD-AXL-Cell-Line
Description	Stable Ba/F3 clone expressing exogenous FLT3-ITD-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+500µg/mL G418
Selection Marker	Puromycin, Neomycin
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 FLT3-ITD-AXL cell Line was generated using retrovirus vector expressing human FLT3-ITD-AXL sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 1µg/mL Puromycin + 500µg/mL G418)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

Quentmeier, H., Reinhardt, J., Zaborski, M. & Drexler, H. G. FLT3-ITD-AXL mutations in acute myeloid leukemia cell lines. Leukemia 17, 120ÿ124 (2003).