KC-4444

293T-rat-NKp46 Cell Line

49121

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Background of 293T-rat-NKp46 Cell Line

NKp46 is also known as NCR1, NCR1 (Natural Cytotoxicity Triggering Receptor 1) is a Protein Coding gene. Diseases associated with NCR1 include Gestational Trophoblastic Neoplasm and Hydatidiform Mole, Recurrent, 1. Among its related pathways are Innate Immune System and Class I MHC mediated antigen processing and presentation. Gene Ontology (GO) annotations related to this gene include obsolete signal transducer activity, downstream of receptor. An important paralog of this gene is LILRA4.

Specifications

Catalog Number	KC-4444
Cell Line Name	293T-rat-NKp46 Cell Line
Host Cell Line	293T
Description	293T cell line stably expressing exogenous rat NKp46 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 + 1 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
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

Cell Line Generation

293t-rat-nkp46 cell line was generated using a lentiviral vector expressing the rat NKp46 sequence.

Characterization

Figure1: Characterization of rat NKp46 overexpression in 293T rat NKp46 stable clone using FACS.

Figure2: Characterization of 293T-rat-NKp46 cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/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 9.0 mL 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 1:4-1:5 every 2-3 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

1.Golden-Mason L, Stone AE, Bambha KM, Cheng L, Rosen HR. Race- and gender-related variation in natural killer p46 expression associated with differential anti-hepatitis C virus immunity. Hepatology. 2012 Oct;56(4):1214-22. doi: 10.1002/hep.25771. PMID: 22505144; PMCID: PMC3458134.
2.Biassoni R, Cantoni C, Marras D, Giron-Michel J, Falco M, Moretta L, Dimasi N. Human natural killer cell receptors: insights into their molecular function and structure. J Cell Mol Med. 2003 Oct-Dec;7(4):376-87. doi: 10.1111/j.1582-4934.2003.tb00240.x. PMID: 14754506; PMCID: PMC6740120.
3.Thorén FB, Riise RE, Ousbäck J, Della Chiesa M, Alsterholm M, Marcenaro E, Pesce S, Prato C, Cantoni C, Bylund J, Moretta L, Moretta A. Human NK Cells induce neutrophil apoptosis via an NKp46- and Fas-dependent mechanism. J Immunol. 2012 Feb 15;188(4):1668-74. doi: 10.4049/jimmunol.1102002. Epub 2012 Jan 9. PMID: 22231698.