KC-1457

293T-NFAT-Luc2 Cell Line

22177

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Background of 293T-NFAT-Luc2 Cell Line

NFAT proteins, which are expressed in most immune-system cells, play a pivotal role in the transcription of cytokine genes and other genes critical for the immune response. Nuclear factor of activated T cells (NFAT), which is the pharmacological target of immunosuppressants cyclosporine and tacrolimus, has been shown to play an important role not only in T cells (immune system), from which their name is derived, but also in many biological events. The activity of NFAT proteins is tightly regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a primary target for inhibition by cyclosporin A and FK506. Calcineurin controls the translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells by interacting with an N-terminal regulatory domain conserved in the NFAT family. The DNA-binding domains of NFAT proteins resemble those of Rel-family proteins, and Rel and NFAT proteins show some overlap in their ability to bind to certain regulatory elements in cytokine genes. NFAT is also notable for its ability to bind cooperatively with transcription factors of the AP-1 (Fos/Jun) family to composite NFAT: AP-1 sites, found in the regulatory regions of many genes that are inducibly transcribed by immune-system cells.

Specifications

Catalog Number	KC-1457
Cell Line Name	293T-NFAT-Luc2 Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of NFAT signaling pathway.
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 +140μg/ml Hygromycin B
Selection Marker	Hygromycin B
Morphology	Epithelial
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

293T-NFAT-Luc2 cell line was generated using lentivirus methods.

Characterization

Figure 1. 293T-NFAT-luciferase cell line was seed into the 96-well plate, and treated with 1uM Ionomycin and 100nM PMA for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 140μg/ml Hygromycin)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 1:4-1:5 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.Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 1997;15:707-47.
2.Kitamura N, Kaminuma O. Isoform-Selective NFAT Inhibitor: Potential Usefulness and Development. Int J Mol Sci. 2021 Mar 8;22(5):2725.
3.Sana I, Mantione ME, Angelillo P, Muzio M. Role of NFAT in Chronic Lymphocytic Leukemia and Other B-Cell Malignancies. Front Oncol. 2021 Apr 1;11:651057.