KC-4161

293T-membrane-IL23-Cell-Line

61374

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Background of 293T-membrane-IL23-Cell-Line

Interleukin-23 (IL-23) is a heterodimeric cytokine belonging to the IL-6/IL-12 family that plays a key role in several of autoimmune and inflammatory disorders. This family contains the 34 type I cytokine receptor chains and 27 ligands, which share structural and functional similarities, but on the other hand they display distinct roles in shaping Th cells responses. IL-12 family cytokines have not only proinflammatory effects but they also promote inflammatory responses. IL-23 is composed of the p40 subunit in common with IL-12, and with a unique p19 subunit. IL-23 binding to an IL-23 receptor expressed on dendritic cells, macrophages and monocytes triggers the activation of Jak2 and Tyk2, which in turn phosphorylates STAT1, STAT3, STAT4 and STAT5 as well as induce formation of STAT3-STAT4 heterodimers. IL-23 is one of the essential factors required for the survival and/or expansion of Th17 cells, which produce IL-17, IL-17F, IL-6 and TNF-α. Th17 cells stimulated by the IL-23 promote osteoclastogenesis through production of IL-17, which induce receptor activator of NF-kappa B ligand on mesenchymal cells. The IL-23-IL-17 axis includes Th17 cells and plays a key role in the development of autoimmune arthritis.

Specifications

Catalog Number	KC-4161
Cell Line Name	293T-membrane-IL23-Cell-Line
NCBI/UniProt Accession Number	P29460 & Q9NPF7
Clone Number	5#
Host Cell Line	293T
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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-membrane-IL23-cell-line was generated using a lentiviral vector expressing the membrane-IL23 sequence.

Characterization

Figure 1: Characterization of membrane-IL23 overexpression in the 293T-membrane-IL23 stable clone using FACS.

Figure 2: Characterization of membrane-IL23 in the 293T-membrane-IL23 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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.Paradowska-Gorycka A, Grzybowska-Kowalczyk A, Wojtecka-Lukasik E, Maslinski S. IL-23 in the pathogenesis of rheumatoid arthritis. Scand J Immunol. 2010 Mar;71(3):134-45.
2.Zhao J, Qi YF, Yu YR. STAT3: A key regulator in liver fibrosis. Ann Hepatol. 2021 Mar-Apr.
3.Zhou L, Wang Y, Wan Q, Wu F, Barbon J, Dunstan R, Gauld S, Konrad M, Leys L, McCarthy R, Namovic M, Nelson C, Overmeyer G, Perron D, Su Z, Wang L, Westmoreland S, Zhang J, Zhu R, Veldman G. A non-clinical comparative study of IL-23 antibodies in psoriasis. MAbs. 2021 Jan-Dec;13(1):1964420.