KC-0138

Ba/F3-cKit-D816V-Cell-Line

19905

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Background of Ba/F3-cKit-D816V-Cell-Line

Mast/stem cell factor receptor (SCFR), also named as proto-oncogene c-Kit or CD117, which is a cytokine receptor mainly expressed on the surface of hematopoietic stem cells, play important roles in gametogenesis, melanogenesis and hematopoiesis. The constitutive activation of C-Kit due to amino-acid mutations, such as D816V/H, V560G, can lead to the anchorage-independent growth and tumorigenicity. 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-0138
Cell Line Name	Ba/F3-cKit-D816V-Cell-Line
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 clone expressing exogenous C-Kit gene bearing D816V 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	Yes

Cell Line Generation

Ba/F3 C-Kit D816V cell line was generated using retrovirus vector expressing human c-Kit sequence bearing D816V mutation.

Characterization

Figure: Characterization of C-Kit and its mutants overexpressing in Ba/F3 stable clones using FACS.

Harvest and seed the Ba/F3 cells expressingC-Kit D816V in 96-well plate (3000 cells/90μL medium).
Next day, add 10μL 10× serially diluted compound solution each well and incubate the plates for another 72 hours.
Add 100μL Cell Titer-Glo each well, mixed and readout using Envision.
Plot the dose-responsive curve and fit the IC₅₀ (the centration of 50% inhibition of DMSO vehicle treated clones) using GraphPad Prism software (Version 5).

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

Mayerhofer, M. et al. Unique effects of KIT D816V in BaF3 cells: induction of cluster formation, histamine synthesis, and early mast cell differentiation antigens. The Journal of Immunology 180, 5466ÿ5476 (2008).
Fletcher, J. A. KIT Oncogenic Mutations: Biologic Insights, Therapeutic Advances, and Future Directions. Cancer Research 76, 6140ÿ6142 (2016).
Kim, S. Y. et al. Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816. Biochemical and Biophysical Research Communications 410, 224ÿ228 (2011).