KC-4023

Jurkat-NFAT-Luc2-CD2-KO-1B4-Cell-Line

40504

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Background of Jurkat-NFAT-Luc2-CD2-KO-1B4-Cell-Line

The glycoprotein CD2 is a costimulatory receptor expressed mainly on T and NK cells that binds to LFA3(CD58), a cell surface protein expressed on e.g., antigen-presenting cells. Its functions are two-fold: adhesion and activation. CD2 serves to facilitate conjugate formation between the T-lineage cell and its cognate partner via intermolecular interaction of CD2 and LFA-3 on the former and latter cells, respectively.CD2 has an important role in the formation and organization of the immunological synapse that is formed between T cells and antigen-presenting cells upon cell-cell conjugation and associated intracellular signaling. CD2 expression is upregulated on memory T cells as well as activated T cells and plays an important role in activation of memory T cells despite the coexistence of several other costimulatory pathways.

Specifications

Catalog Number	KC-4023
Cell Line Name	Jurkat-NFAT-Luc2-CD2-KO-1B4-Cell-Line
Host Cell Line	Jurkat-NFAT-Luc2-reporter
Description	Stable Jurkat-NFAT-Luc2-reporter clone with human CD2 gene knockout, No.1B4
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS+300μg/mL Hygromycin B
Selection Marker	N/A
Morphology	lymphoblast
Subculture	Split saturated culture 1:5-1:8 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 26 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Jurkat-NFAT-Luc2-CD2-KO-1B4 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of Jurkat-NFAT-Luc2-CD2-KO-1B4 cell line stable clone using PCR sequencing.

Figure 2: Characterization of Jurkat-NFAT-Luc2-CD2-KO-1B4 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of Jurkat-NFAT-Luc2-CD2-KO-1B4 cell line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS+300μg/mL Hygromycin B)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 1:5-1:8 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

Binder C, Cvetkovski F, Sellberg F, Berg S, Paternina Visbal H, Sachs DH, Berglund E, Berglund D. CD2 Immunobiology. Front Immunol. 2020 Jun 9;11:1090. doi: 10.3389/fimmu.2020.01090. PMID: 32582179; PMCID: PMC7295915.
Moingeon P, Chang HC, Sayre PH, Clayton LK, Alcover A, Gardner P, Reinherz EL. The structural biology of CD2. Immunol Rev. 1989 Oct;111:111-44. doi: 10.1111/j.1600-065x.1989.tb00544.x. PMID: 2576417.