KC-0294

293T-cyno-DLL3-Cell-Line

20179

Home » 293T-cyno-DLL3-Cell-Line

Background of 293T-cyno-DLL3-Cell-Line

Delta-Like 3，abbreviated as DLL3，is a member of ligand protein family, functioned as Notch ligand, and play a important role in somitogensis. DLL3 is also overexpression in neuroendocrine tumors, which has been identified as a potential target for tumor therapy. Rovapituzumab tesirine, which targeted DLL3, have been used as experimental treatment of lung cancer.

Specifications

Catalog Number	KC-0294
Cell Line Name	293T-cyno-DLL3-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	HEK293T cell line stable expressing exogenous cynomolgus DLL3 gene
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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+1µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T Cynomolgus DLL3 cell line was generated using lentiviral vector expressing cynomolgus DLL3 sequence.

Characterization

Figure: Characterization of cyno DLL3 overexpressing in 293T stable clones using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 1μg/mL puromycin)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 1:3-1:6 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Turnpenny, P. D. et al. A gene for autosomal recessive spondylocostal dysostosis maps to 19q13.1-q13.3. The American Journal of Human Genetics 65, 175ÿ182 (1999).
Chapman, G., Sparrow, D. B., Kremmer, E. & Dunwoodie, S. L. Notch inhibition by the ligand Delta-Like 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis. Human Molecular Genetics 20, 905ÿ916 (2010).
Saunders, L. R. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Science Translational Medicine 7, 302ra136ÿ302ra136 (2015).