KC-1510

Jurkat-NFAT-Luc2-LAG3-CD3zeta

22281

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Background of Jurkat-NFAT-Luc2-LAG3-CD3zeta

"Lymphocyte-activation gene 3(LAG3), also known as CD223, is a member of immunoglobulin superfamily, expressed on activated T cells, NK cells, B cells and DC cells. LAG3 can bind to MHC II molecules and induce maturation of DC cells, and cytokine secretion of cytotoxic T cells and NK cells. LAG3 is a promising drug target of cancer therapy and autoimmune disease, its therapeutic antibodies including BMS-986016 and GSK2831781 had already been used in the clinical trial. 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-1510
Cell Line Name	Jurkat-NFAT-Luc2-LAG3-CD3zeta
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 LAG3-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 + 300μg/mL Hygromycin + 0.75μg/mL puromycin
Selection Marker	Hygromycin B and puromycin
Morphology	Lymphoblast
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1 × 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-LAG3-CD3zeta cell line was generated using lentivirus vector expressing LAG3-CD3zeta fusion sequence.

Characterization

Figure 1: Characterization of human LAG3 overexpression in Jurkat-NFAT-Luc2-LAG3-CD3zeta stable clones using FCAS.

Figure 2: Jurkat-NFAT-Luc2-LAG3-CD3zeta cells were seeded into the 96-well plate, treated with Daudi for 16 hours, and then readout using Bright-Glo Detection System.

Figure 3: Jurkat-NFAT-Luc2-LAG3-CD3zeta cells were seeded into the 96-well plate, treated with Lag3 mAb for 16 hours, and then readout using Bright-Glo Detection System.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 300μg/mL Hygromycin B and 0.75μg/mL puromycin).
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 (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

Anderson, Ana C, Nicole Joller, and Vijay K Kuchroo. 2016. “Lag-3, Tim-3, and TIGIT: Co-Inhibitory Receptors with Specialized Functions in Immune Regulation.” Immunity 44 (5). Elsevier Inc.: 989–1004.
Huard, B, R Mastrangeli, P Prigent, D Bruniquel, S Donini, N El-Tayar, B Maigret, M Dréano, and F Triebel. 1997. “Characterization of the Major Histocompatibility Complex Class II Binding Site on LAG-3 Protein.” Proceedings of the National Academy of Sciences 94 (11): 5744–49.