KC-2356

CHOK1-CLDN3 Cell Line

23896

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Background of CHOK1-CLDN3 Cell Line

CLDN3, also known as claudin-3, is a transmembrane protein belonging to claudin family, which is essential for the formation and maintenance of epithelial tight junctions. Abnormal expression of claudin-3 may lead to structural and functional damage of tight junctions, which plays an important part in tumorigenesis and cancer progression.

Specifications

Catalog Number	KC-2356
Cell Line Name	CHOK1-CLDN3 Cell Line
Clone Number	1#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous CLDN3 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-2 × 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

CHOK1-CLDN3 Cell Line was generated using a lentiviral vector expressing the CLDN3 sequence.

Characterization

Figure 1: Characterization of CLDN3 overexpression in the CHO-K1 CLDN3 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 10μg/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 1:4-1:5 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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.Kosuke Yamaga. Claudin-3 Loss Causes Leakage of Sweat from the Sweat Gland to Contribute to the Pathogenesis of Atopic Dermatitis. J Invest Dermatol. 2018 Jun;138(6):1279-1287.
2. N V Danilova , K A Anikina & N A Oleynikova. Claudin-3 expression in gastric cancer. Arkh Patol. 2020;82(2):5- 11.