KC-4476

CHOK1 rat NKP46 Middle Cell Line

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Background of CHOK1 rat NKP46 Middle Cell Line

The NKp46 protein was named as such since a 46-kDa band was precipitated from NK cells, using NKp46-specific mAb. Molecular cloning of the NKp46 cDNA revealed that NKp46 is a member of the immunoglobulin superfamily, characterized by two extracellular C2-type Ig-like domains.Although it has been recently demonstrated that NKp46 is expressed on a special subset of lymphocytes of the innate immune system, NKp46 is still considered a unique NK cell marker and is the only NCR that has a mouse ortholog, named NCR1.

Specifications

Catalog Number	KC-4476
Cell Line Name	CHOK1 rat NKP46 Middle Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous rat NKP46 gene in middle level
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 1-3 × 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

CHOK1 rat NKP46 Cell Line was generated using a lentiviral vector expressing the rat NKP46 sequence.

Characterization

Figure 1: Characterization of rat NKP46 overexpression in the CHO-K1 rat NKP46 stable clone using FCAS.

Figure 2: Characterization of rat NKP46 overexpression in the CHO-K1 rat NKP46 stable clone using PCR sequence.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Glasner A,Ghadially H, Gur C, et al. Recognition and prevention of tumor metastasis by the NK receptor NKp46/NCR1. J Immunol. 2012 Mar 15;188(6):2509-15. doi: 10.4049/jimmunol.1102461.
Mandelboim O, Porgador A. NKp46.Int J Biochem Cell Biol. 2001 Dec;33(12):1147-50. doi: 10.1016/s1357-2725(01)00078-4.