KC-1257

293T-CLDN18.1-Flag-Cell-Line

21807

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Background of 293T-CLDN18.1-Flag-Cell-Line

CLDN18.1 is the isoform 1 of the claudin 18 gene, which belong to the lager claudin family and play the important role in cell tight junction in epithelial cells. The other isoform, CLND18.2, is found overexpressed on gastrointestinal adenocarcinoma and pancreatic tumors. The identification as a tumor target of CLDN18.2 has led to the repaid progress of antibody treatment of gastrointestinal adenocarcinoma and pancreatic tumors, such as IMAB362 (Claudiximab).

Specifications

Catalog Number	KC-1257
Cell Line Name	293T-CLDN18.1-Flag-Cell-Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous CLDN18.1 and Flag tag 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-CLDN18.1-Flag cell line was generated using lentiviral vector expressing CLDN18.1 and Flag tag sequence.

Characterization

Figure: Characterization of Flag tag overexpressing in 293T-CLDN18.1-Flag stable clones using intracellular 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

Micke, Patrick, Johanna Sofia Margareta Mattsson, Karolina Edlund, Miriam Lohr, Karin Jirstr?m, Anders Berglund, Johan Botling, et al. 2014. ¡ùAberrantly Activated Claudin 6 and 18.2 as Potential Therapy Targets in Non-Small-Cell Lung Cancer.¡ì International Journal of Cancer 135 (9): 2206Ã¿14. doi:10.1002/ijc.28857.
Singh, Prabhsimranjot, Sudhamshi Toom, and Yiwu Huang. 2017. ¡ùAnti-Claudin 18.2 Antibody as New Targeted Therapy for Advanced Gastric Cancer,¡ì May. Journal of Hematology & Oncology, 1Ã¿5. doi:10.1186/s13045-017-0473-4.
Sahin, U, M Koslowski, K Dhaene, D Usener, G Brandenburg, G Seitz, C Huber, and O Tureci. 2008. ¡ùClaudin-18 Splice Variant 2 Is a Pan-Cancer Target Suitable for Therapeutic Antibody Development.¡ì Clinical Cancer Research 14 (23): 7624Ã¿34. doi:10.1158/1078-0432.CCR-08-1547.