KC-1675

Ba/F3-CCDC6-RET-Cell-Line

22593

Home » 细胞系 » Ba/F3-CCDC6-RET-Cell-Line

Background of Ba/F3-CCDC6-RET-Cell-Line

RET, abbreviated for "rearranged during transfection" is a receptor tyrosine kinase for membranes of the gial cell line-derived neurotropic neurotrophic factor (GDNF) family of extracellular signaling molecules. Overactivation of RET have associated with a number of cancers. The identification of RET as a driver gene has led to the development of anticancer therapeutics agents. 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-1675
Cell Line Name	Ba/F3-CCDC6-RET-Cell-Line
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 clone expressing exogenous CCDC6-RET fusion protein.
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 CCDC6-RET cell line was generated using retrovirus vector expressing human CCDC6-RET sequence.

Characterization

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

Kawamoto Y, Takeda K, Okuno Y, et al. (2004). "Identification of RET autophosphorylation sites by mass spectrometry". J. Biol. Chem. 279 (14): 14213Ã¿24.
Rudin, Charles M, Alexander Drilon, and J T Poirier. 2014. ¡ùRET Mutations in Neuroendocrine Tumors: Including Small-Cell Lung Cancer.¡ì Journal of Thoracic Oncology 9 (9). Elsevier: 1240Ã¿42.
Kohno, Takashi, Koji Tsuta, Katsuya Tsuchihara, Takashi Nakaoku, Kiyotaka Yoh, and Koichi Goto. 2013. ¡ùRET Fusion Gene: Translation to Personalized Lung Cancer Therapy.¡ì Cancer Science 104 (11): 1396Ã¿1400.