KC-3288

Ba/F3-cMET Cell Line

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Background

c-Met, also named as hepatocyte growth factor receptor (HGFR), is a single pass tyrosine kinase receptor, which is mainly expressed on the cells of epithelial origin, and play essential role in embryonic development, organogenesis and wound healing. HGF and its splicing isoform are the only know ligands of cMet. Abnormal activation of cMet due to overexpression of Met or its ligands, fusion mutation as well as exon14 skipping have been implicated in oncogenesis. 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-3288
Cell Line Name:
Ba/F3 Met Cell Line
Price:
0
Host Cell Line:
Mouse Ba/F3 cell line
Description:
Stable Ba/F3 clone expressing exogenous Met sequence.
Quantity:
One vial of frozen cells (5X106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% RPMI1640+20% FBS+10% DMSO
Propagation Medium:
RPMI1640+10%FBS+8ng/mL mouse IL-3+1µg/mL Puromycin
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 c-Met cell line was generated using a lentiviral vector expressing the human c-Met sequence.

Characterization

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Application

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Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 8ng/mL mouse IL-3 + 1µg/mL Puromycin) 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% 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

References

1. Saffroy, R. et al. MET exon 14 mutations as targets in routine molecular analysis of primary sarcomatoid carcinoma of the lung. Oncotarget 8, 42428–42437 (2017). 2. Peschard, P. & Park, M. From Tpr-Met to Met, tumorigenesis and tubes. Oncogene 26, 1276–1285 (2007).
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