KC-1937

CT26.WT-human-CD94-NKG2A Cell Line

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Background of CT26.WT-human-CD94-NKG2A 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. NKG2A 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.

Specifications

Catalog Number	KC-1937
Cell Line Name	CT26.WT-human-CD94-NKG2A Cell Line
Host Cell Line	CT26.WT
Description	CT26.WT cell line stable expressing exogenous human CD94 and 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

CT26.WT human CD94 NKG2A Cell Line was generated using a lentiviral vector expressing the CD94 and NKG2A sequence.

Characterization

Figure 1: Characterization of CD94 and NKG2A overexpression in the CT26.WT human CD94 NKG2A stable clone using FACS.

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

Turnpenny, P. D. et al. A gene for autosomal recessive spondylocostal dysostosis maps to 19q13.1-q13.3. The American Journal of Human Genetics 65, 175–182 (1999).
Chapman, G., Sparrow, D. B., Kremmer, E. & Dunwoodie, S. L. Notch inhibition by the ligand Delta-Like 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis. Human Molecular Genetics 20, 905–916 (2010).
Saunders, L. R. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Science Translational Medicine 7, 302ra136–302ra136 (2015).