KC-1981

293T-cyno-TIGIT-Cell-Line

23171

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Background of 293T-cyno-TIGIT-Cell-Line

TIGIT (T cell Immunoreceptor with Ig and ITIM domains), also called Vstm3 (V-set and transmembrane domain-containing 3), Vsig9 (V-set and Ig domain-containing 9) and WUCAM (Washington University cell adhesion molecule) is a type I transmembrane protein belonging to B7-CD28 superfamily. TIGIT is mainly expressed on NK cells and activated, memory and regulatory T cells. It can bind to CD155 and CD122, and play an important role in T cell activation and susceptibility to autoimmune challenges.

Specifications

Catalog Number	KC-1981
Cell Line Name	293T-cyno-TIGIT-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable HEK293T cell line expressing exogenous monkey TIGIT 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 cyno TIGIT cell line was generated using a lentiviral vector expressing the cyno TIGIT 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

Anderson, Ana C, Nicole Joller, and Vijay K Kuchroo. 2016. ¡ùLag-3, Tim-3, and TIGIT: Co-Inhibitory Receptors with Specialized Functions in Immune Regulation.¡ì Immunity 44 (5). Elsevier Inc.: 989Ã¿1004. doi:10.1016/j.immuni.2016.05.001.
Chauvin, Joe-Marc, Ornella Pagliano, Julien Fourcade, Zhaojun Sun, Hong Wang, Cindy Sander, John M Kirkwood, et al. 2015. ¡ùTIGIT and PD-1 Impair Tumor AntigenÃ¿Specific CD8+ T Cells in Melanoma Patients¡ì 125 (5): 2046Ã¿58. doi:10.1172/JCI80445.