KC-1503

Jurkat-NFAT-Luc2-PD1 Cell Line

22267

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Background of Jurkat-NFAT-Luc2-PD1 Cell Line

PD-1, human programmed cell death 1 protein, is a receptor belonging to the immunoglobulin superfamily which is expressed primarily on activated T cells. NK cells, B cells, and monocytes. PD-1 interaction with its ligands, PD-L1 and PD-L2, leads to the downregulation of T cell responses, including T cell proliferation and cytokine production, and limits immune destruction of tissues. The interaction between PD-1 and its ligands, thus plays a role in maintaining the balance between immune activation and tolerance, potentially including tumor tolerance. NFAT (nuclear factor of activator T cells) proteins belong 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 the T cell receptor (TCR).

Specifications

Catalog Number	KC-1503
Cell Line Name	Jurkat-NFAT-Luc2-PD1 Cell Line
Host Cell Line	Jurkat-NFAT-Luc2
Description	Jurkat cell line stable expressing exogenous luciferase gene under the control of NFAT responsive element and full-length human PD1 protein.
Quantity	One vial 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+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-PD1 cell line was generated using lentiviral vector expressing full-length sequence.

Characterization

Figure 1. Characterization of PD1 overexpression in Jurkat-NFAT-Luc2-PD1 reporter cell line.

Figure 2. Jurkat-NFAT-Luc2-PD1 cells and KC-1148 293T-OS8-PDL1 cells were seeded into 96-well plates, treated with different antibodies for 6 hours, and then read out using Bright-Glo Detection System.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 300μg/ml Hygromycin 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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% 1640 + 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

Boussiotis, V. A. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med 375, 1767– 1778 (2016).

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.