KC-1639

Jurkat-NFAT-Luc2-TIGHT-CD3zeta Cell Line

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Background of Jurkat-NFAT-Luc2-TIGHT-CD3zeta Cell Line

T cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed on lymphocytes. TIGIT interacts with CD155 expressed on antigen-presenting cells or tumour cells to down-regulate T cell and natural killer (NK) cell functions. TIGIT has emerged as a key inhibitor of anti-tumour responses that can hinder multiple steps of the cancer immunity cycle. Pre-clinical studies indicated that TIGIT blockade may protect against various solid and haematological cancers. Several monoclonal antibodies (mAbs) that block the inhibitory activity of human TIGIT have been developed. CD247, which is also known as CD3ζ, CD3H, CD3Q, CD3Z, IMD25, T3Z and TCRZ, encodes CD3ζ protein, which is expressed primarily in natural killer (NK) and T cells. SLE is associated with a deficiency in cluster of differentiation 247 (CD247, also known as CD3 zeta chain), a component of the T-cell receptor (TCR)-CD3 complex. A comprehensive analysis showed that in more than half of SLE patients tested CD247 expression was either attenuated or absent.

Specifications

Catalog Number	KC-1639
Cell Line Name	Jurkat-NFAT-Luc2-TIGHT-CD3zeta Cell Line
Host Cell Line	Jurkat-NFAT-Luc2
Description	Stable Jurkat-NFAT-Luc2 cell line expressing exogenous TIGIT-CD3Zeta fusion 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 +300ug/ml Hygromycin B+0.75ug/ml Puromycin
Selection Marker	Puromycin, Hygromycin B
Morphology	lymphoblast
Subculture	Split saturated culture 1:4-1:5 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

Jurkat-NFAT-Luc2-TIGHT-CD3zeta Cell Line was generated using a lentiviral vector expressing TIGIT-CD3Zeta sequence.

Characterization

Figure 1. Characterization of human TIGIT overexpression in Jurkat-NFAT-Luc2-TIGHT-CD3zeta cell line clone using FCAS.

Figure 2. Stimulation effect of Jurkat-NFAT-Luc2-TIGHT-CD3zeta cell line co-cultured with 293T or 293T-CD155 cells for 6h.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10%FBS, 300ug/ml Hygromycin B and 0.75µg/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 (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

1.Harjunpää H, Guillerey C. TIGIT as an emerging immune checkpoint. Clin Exp Immunol. 2020 May;200(2):108-119. doi: 10.1111/cei.13407. Epub 2019 Dec 25. PMID: 31828774; PMCID: PMC7160651.
2.Dexiu C, Xianying L, Yingchun H, Jiafu L. Advances in CD247. Scand J Immunol. 2022 Jul;96(1):e13170. doi: 10.1111/sji.13170. Epub 2022 Apr 17. PMID: 35388926.
3.Dexiu C, Xianying L, Yingchun H, Jiafu L. Advances in CD247. Scand J Immunol. 2022 Jul;96(1):e13170. doi: 10.1111/sji.13170. Epub 2022 Apr 17. PMID: 35388926.