KC-4681

Jurkat-TNFα-R78T-S79T Cell Line

57673

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Background of Jurkat-TNFα-R78T-S79T Cell Line

This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, psoriasis, rheumatoid arthritis ankylosing spondylitis, tuberculosis, autosomal dominant polycystic kidney disease, and cancer. Mutations in this gene affect susceptibility to cerebral malaria, septic shock, and Alzheimer disease. Knockout studies in mice also suggested the neuroprotective function of this cytokine.

Specifications

Catalog Number	KC-4681
Cell Line Name	Jurkat-TNFα-R78T-S79T Cell Line
Clone Number	4-9#
Host Cell Line	Jurkat
Description	Stable Jurkat cell line expressing exogenous TNFα protein with R78T-S79T mutation.
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	RPMI 1640 + 10% FBS + 1ug/mL Puromycin
Selection Marker	Puromycin
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-TNFα-R78T-S79T cell line was generated using a lentiviral vector expressing the TNFα-R78T-S79T sequence.

Characterization

Figure 1: Characterization of TNFα-R78T-S79T overexpression in the Jurkat stable clone using FACS.

Figure 2: Characterization of TNFα-R78T-S79T mutant overexpressing in the Jurkat stable clone by using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI 1640 + 10% FBS + 1ug/mL Puromycin )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% 1640 + 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. Jinesh G G, Chunduru S, Kamat AM. Smac mimetic enables the anticancer action of BCG-stimulated neutrophils through TNF-α but not through TRAIL and FasL. J Leukoc Biol. 2012 Jul;92(1):233-44. doi: 10.1189/jlb.1211623. Epub 2012 Apr 18. PMID: 22517918; PMCID: PMC3382315.
2. Nie H, Zheng Y, Li R, Guo TB, He D, Fang L, Liu X, Xiao L, Chen X, Wan B, Chin YE, Zhang JZ. Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-α in rheumatoid arthritis. Nat Med. 2013 Mar;19(3):322-8. doi: 10.1038/nm.3085. Epub 2013 Feb 10. PMID: 23396208.