KC-6256

Hepa1-6-mouse-NK1.1 Cell Line

60660

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Background of Hepa1-6-mouse-NK1.1 Cell Line

Natural killer (NK) cells are lymphocytes that mediate cytotoxicity and secrete cytokines after immune stimulation. Several genes of the C-type lectin superfamily, including the rodent NKRP1 family of glycoproteins, are expressed by NK cells and may be involved in the regulation of NK cell function. The KLRB1 protein contains an extracellular domain with several motifs characteristic of C-type lectins, a transmembrane domain, and a cytoplasmic domain. The KLRB1 protein is classified as a type II membrane protein because it has an external C terminus. KLRB1c Enables identical protein binding activity and signaling receptor activity. Acts upstream of or within natural killer cell activation and positive regulation of natural killer cell mediated cytotoxicity. Located in external side of plasma membrane. Is expressed in thymus primordium. Orthologous to human KLRB1 (killer cell lectin like receptor B1).

Specifications

Catalog Number	KC-6256
Cell Line Name	Hepa1-6-mouse-NK1.1 Cell Line
NCBI/UniProt Accession Number	NM_001159904
Clone Number	2#
Host Cell Line	Hepa1-6
Description	Hepa1-6 cell line stably expressing exogenous mouse-NK1.1 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 + 2 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:4 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

Cell Line Generation

Hepa1-6-mouse-NK1.1 cell line was generated using a lentiviral vector expressing the mouse-NK1.1 sequence.

Characterization

Figure1: Characterization of mouse-NK1.1 overexpressing in Hepa1-6 stable clones using FACS.

Figure2: Characterization of Hepa1-6-mouse-NK1.1 cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 2 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 9.0mL 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 1:2-1:4 every 2-3 days; seed out at about 1-2 × 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

Stenström M, Sköld M, Andersson A, Cardell SL. Natural killer T-cell populations in C57BL/6 and NK1.1 congenic BALB.NK mice-a novel thymic subset defined in BALB.NK mice. Immunology. 2005 Mar;114(3):336-45. doi: 10.1111/j.1365-2567.2004.02111.x. PMID: 15720435; PMCID: PMC1782094.
Glimcher L, Shen FW, Cantor H. Identification of a cell-surface antigen selectively expressed on the natural killer cell. J Exp Med. 1977 Jan 1;145(1):1-9. doi: 10.1084/jem.145.1.1. PMID: 187714; PMCID: PMC2180593.
Asjö B, Kiessling R, Klein G, Povey S. Genetic variation in antibody response and natural killer cell activity against a Moloney virus-induced lymphoma (YAC). Eur J Immunol. 1977 Aug;7(8):554-8. doi: 10.1002/eji.1830070812. PMID: 902682.