KC-1637

Ba/F3-TPR-Met-F1200I-Cell-Line

22517

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Background of Ba/F3-TPR-Met-F1200I-Cell-Line

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. This is because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of the IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-1637
Cell Line Name	Ba/F3-TPR-Met-F1200I-Cell-Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous Met-F1200I mutation with TPR fusion .
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	NA

Cell Line Generation

Ba/F3-TPR-Met-F1200I cell Line was generated using a retrovirus vector expressing the human Met-F1200I sequence with TPR fusion.

Characterization

Figure: Characterization of Met and its mutants overexpressing in Ba/F3 stable clones using western blot.

a. Cell-based kinase inhibition screening b. Cell viability assay c. In vivo efficacy study Example: kinase inhibitors screening 1. Harvest and seed the Ba/F3 cells expressing the MET mutant in a 96-well plate (3000 cells/90ul medium). 2. Add 10ul 10X serially diluted compound solution to each well the following day and incubate the plate for another 72 hours. 3. Add 100ul Cell Titer-Glo to each well, mix well and perform the analysis using Envision. 4. Plot the dose-responsive curve and fit the IC50 value (the centration of 50% inhibition of DMSO vehicle treated clones) using the GraphPad Prism software (Version 7).

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 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

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).
Peschard, P. & Park, M. From Tpr-Met to Met, tumorigenesi.s and tubes. Oncogene 26, 127&-1285 (2007).