KC-1458

Jurkat-NFAT-Luc2 Cell Line

26431

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Background of Jurkat-NFAT-Luc2 Cell Line

NFAT (nuclear factor of activator T cells) proteins belongs to a family of transcription factors that are very important in T cells activation, differentiation and tolerance. NFAT pathway is activated by Ca2+ and the Ca2+/calmodulin dependent serine phosphatase calcineurin after the engagement of T cell receptor (TCR).

Specifications

Catalog Number	KC-1458
Cell Line Name	Jurkat-NFAT-Luc2 Cell Line
Host Cell Line	Jurkat
Description	Jurkat cell line stable expressing exogenous luciferase gene under the control of NFAT responsive element.
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
Selection Marker	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 cell line was generated using lentivirus method.

Characterization

Figure 1. Activation of Jurkat-NFAT-Luc2 cell line stimulated with CD3/CD28 antibodies or PMA and ionomycin. 20,000 Jurkat-NFAT-Luc2 cell line was seeded into the 96-well plate, and treated with differentstimulus, then readout with Bright-Glo system.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 300μg/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% 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.Macian, F. NFAT proteins: Key regulators of T-cell development and function. Nat. Rev. Immunol. 5, 472–484 (2005).
2.Hogan, P. G., Chen, L., Nardone, J. & Rao, A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 17, 2205–32 (2003).