KC-1986

HEK293T-NKG2A-Cell-Line

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Background of HEK293T-NKG2A-Cell-Line

NKG2, also named as KLRC1 or CD159a, is a type II transmembrane protein predominately expressed in NK cells. NKG2 has two isoforms, NKG2A and NKG2B, both of which contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the intracellular part of the molecule, NKG2A can form heterodimers with CD94, linked by disulfide bonds and play an inhibitory role in immunological process after recognizes non-classical major histocompatibility (MHC) class Ib molecule HLA-E loaded with self-peptides derived from the signal sequence of classical MHC class Ia molecules.

Specifications

Catalog Number	KC-1986
Cell Line Name	HEK293T-NKG2A-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable HEK293T cell line expressing exogenous human NKG2A 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 human NKG2A cell line was generated using a lentiviral vector expressing the human NKG2A sequence.

Characterization

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

Borrego, F., Masilamani, M., Marusina, A. I., Tang, X. & Coligan, J. E. The CD94/NKG2 family of receptors: from molecules and cells to clinical relevance. Immunol Res 35, 263ÿ278 (2006).
Gunturi, A., Berg, R. E. & Forman, J. The role of CD94/NKG2 in innate and adaptive immunity. Immunol Res 30, 29ÿ34 (2004).