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.
Cell Line Name:
Ba/F3 C-Kit-V559D-Y823D Cell Line
Host Cell Line:
Mouse Ba/F3 cell line
Stable Ba/F3 clone expressing exogenous c-Kit protein bearing V559D and Y823D double mutations
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% RPMI1640+20% FBS+10% DMSO
Mostly single, round (some polymorph) cells in suspension
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 20 hours
Cell Line Generation
Ba/F3 cKIT-V559D-Y823D cell Line was generated using a retrovirus vector expressing the human cKIT-V559D-Y823D sequence.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (RPMI1640 + 10%FBS) 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.
1. Prepare the freezing medium (70% RPMI-1640 + 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
1. 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, 5466–5476 (2008).
2. Fletcher, J. A. KIT Oncogenic Mutations: Biologic Insights, Therapeutic Advances, and Future Directions. Cancer Research 76, 6140–6142 (2016).
3. 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, 224–228 (2011).
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