KC-4692

Jurkat-NFAT-Luc2-CD28-KO-3A4 Cell Line

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Background of Jurkat-NFAT-Luc2-CD28-KO-3A4 Cell Line

The CD28 gene, located on chromosome 2q33, encodes a protein that is a key co-stimulatory molecule in T-cell activation. CD28 is expressed on the surface of T-cells and interacts with B7 molecules (CD80 and CD86) on antigen-presenting cells (APCs). This interaction is essential for the full activation of T-cells, leading to their proliferation, cytokine production, and differentiation into effector and memory cells. Without this co-stimulation, T-cells can become anergic or undergo apoptosis.The importance of CD28 in immune responses is well-documented. It plays a crucial role in maintaining the balance between immune activation and tolerance. Dysregulation of CD28 signaling has been linked to various autoimmune and inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Additionally, CD28 is a target for immunotherapy, particularly in the context of cancer treatment. Chimeric antigen receptor (CAR) T-cells, which are engineered to express CD28 as part of their co-stimulatory domain, have shown significant promise in treating hematological malignancies.

Specifications

Catalog Number	KC-4692
Cell Line Name	Jurkat-NFAT-Luc2-CD28-KO-3A4 Cell Line
Clone Number	3A4
Host Cell Line	Jurkat-NFAT-Luc2
Description	Stable Jurkat-NFAT-Luc2 clone with human CD28 gene knockout, No.3A4
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	Hygromycin B
Morphology	Lymphoblast
Subculture	Split saturated culture 1:4-1:6 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

Cell Line Generation

Jurkat-NFAT-Luc2-CD28-KO-3A4 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of Jurkat-NFAT-Luc2-CD28-KO-3A4 cell line stable clone using PCR sequencing.

Figure 2: Characterization of Jurkat-NFAT-Luc2-CD28-KO-3A4 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of Jurkat-NFAT-Luc2-CD28-KO-3A4 cell line stable clone using FACS.

Figure 4: Jurkat-NFAT-Luc2-CD28-KO and Jurkat-NFAT-Luc2 cell line were seeded into the 96-well plate, and treated with Theralizumab(Cat#KB-1158, Kyinno) at a maximum concentration of 20μg/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640+10% FBS+300μg/mL Hygromycin B)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.June CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med. 2018 Jul 5;379(1):64-73. doi: 10.1056/NEJMra1706169. PMID: 29972754; PMCID: PMC7433347.
2.Linsley PS, Brady W, Grosmaire L, Aruffo A, Damle NK, Ledbetter JA. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J Exp Med. 1991 Mar 1;173(3):721-30. doi: 10.1084/jem.173.3.721. PMID: 1847722; PMCID: PMC2118836.
3.Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol. 2002 Feb;2(2):116-26. doi: 10.1038/nri727. PMID: 11910893.