KC-1951

293T-CD94-NKG2C-Cell-Line

23111

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Background of 293T-CD94-NKG2C-Cell-Line

CD94 & NKG2A, also known as KLRD1 & CD159A. CD94 is a type II integral membrane protein belonging to lectin superfamily, which is plays a role as a receptor for the recognition of MHC class I Human Leukocyte Antigen (HLA)-E molecules by NK cells and some cytotoxic T-cells.NKG2-A contains C-type lectin domain and belongs to the killer cell lectin-like receptor (KLR) family. KLR family is a group of transmembrane proteins preferentially expressed in natural killer (NK) cells. CD94 can form disulfide-bonded heterodimer with NKG2A on the surface of NK cells. The CD94/NKG2A complex interacts with HLA-E on target cells and inhibit the cytotoxic activity of NK cells to prevent cell lysis. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-1951
Cell Line Name	293T-CD94-NKG2C-Cell-Line
Host Cell Line	Human 293T cell line
Description	Stable 293T cell line expressing exogenous human CD94 and NKG2C 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 CD94 NKG2C cell line was generated using a lentiviral vector expressing the human CD94 and NKG2C 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 AI, Tang X, Coligan JE (2006), The CD94/NKG2 family of receptors: from molecules and cells to clinical relevance. Immunol Res. 35(3):263-78
CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NKcell subsets. Jianhua Yu, Hsiaoyin C. Mao, Min Wei, Tiffany Hughes, Jianying Zhang, Il-kyoo Park, Shujun Liu, Susan McClory, Guido Marcucci, Rossana Trotta, Michael A. CaligiuriBlood (2010) 115 (2): 274ÿ281.
Borrego F, Kabat J, Kim DK, Lieto L, Maasho K, Pena J et al. Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol Immunol 2002; 38: 637ÿ660.