KC-2977

293T-CLEC2D-Cell-Line

26845

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Background of 293T-CLEC2D-Cell-Line

CLEC2D encodes a member of the natural killer cell receptor C-type lectin family. The encoded protein inhibits osteoclast formation and contains a transmembrane domain near the N-terminus as well as the C-type lectin-like extracellular domain. Several alternatively spliced transcript variants have been identified for this gene.

Specifications

Catalog Number	KC-2977
Cell Line Name	293T-CLEC2D-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CLEC2D 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 1:4-1:8 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

293T-CLEC2D-cell-line was generated using a lentiviral vector expressing the CLEC2D sequence.

Characterization

Figure 1: Characterization of CLEC2D overexpression in the 293T-CLEC2D stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 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 1:4-1:8 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.Santos-Juanes J, Fernández-Vega I, Lorenzo-Herrero S, Sordo-Bahamonde C, Martínez-Camblor P, García-Pedrero JM, Vivanco B, Galache-Osuna C, Vazquez-Lopez F, Gonzalez S, Rodrigo JP. Lectin-like transcript 1 (LLT1) expression is associated with nodal metastasis in patients with head and neck cutaneous squamous cell carcinoma. Arch Dermatol Res. 2019 Jul;311(5):369-376. doi: 10.1007/s00403-019-01916-x. Epub 2019 Apr 6. PMID: 30955082.
Mathew PA, Chuang SS, Vaidya SV, Kumaresan PR, Boles KS, Pham HT. The LLT1 receptor induces IFN-gamma production by human natural killer cells. Mol Immunol. 2004 Mar;40(16):1157-63. doi: 10.1016/j.molimm.2003.11.024. PMID: 15104121.