KC-5366

293T-IL12RB1-IL23R Cell Line

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Background of 293T-IL12RB1-IL23R Cell Line

Human IL12RB1 encodes IL12Rβ1, a type I transmembrane receptor that is an essential component of the IL12- and IL23-signaling complex. IL12RB1 is well-established as being a promoter of delayed type hypersensitivity (DTH), the immunological reaction that limits tuberculosis. Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rβ1 as a receptor signaling subunit. Mutations in this gene impair the development of interleukin-17-producing T lymphocytes and result in increased susceptibility to mycobacterial and Salmonella infections.
Interleukin (IL-)23 is a central cytokine controlling TH17 development. Overshooting IL-23 signaling contribute to autoimmune diseases. IL-23 belongs to the IL-12 cytokine family, which is produced by activated dendritic cells (DC) and macrophages. IL-23 acts by binding to its receptor consisting of two distinct subunits, IL-12Rβ1 and IL-23R. This, in turn, leads to janus kinase (JAK) activation and signal transducer and activator of transcription (STAT) 3/4 phosphorylation.

Specifications

Catalog Number	KC-5366
Cell Line Name	293T-IL12RB1-IL23R Cell Line
NCBI/UniProt Accession Number	NM_005535.3/XM_011540790.4
Clone Number	4-3#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human IL12RB1 and IL23R 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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-IL12RB1-IL23R cell line was generated using a lentiviral vector expressing the human IL12RB1 and IL23R sequence.

Characterization

Figure 1: Characterization of human IL12Rβ1 and IL23R overexpression in the 293T-IL12RB1-IL23R stable clone using FACS.

Figure 2: Characterization of human IL12RB1 and IL23R in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 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 1:4-1:5 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. Robinson RT. IL12Rβ1: the cytokine receptor that we used to know. Cytokine. 2015 Feb;71(2):348-59. doi: 10.1016/j.cyto.2014.11.018. Epub 2014 Dec 13. PMID: 25516297; PMCID: PMC4346141.
2. Glassman CR, Mathiharan YK, Jude KM, Su L, Panova O, Lupardus PJ, Spangler JB, Ely LK, Thomas C, Skiniotis G, Garcia KC. Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells. Cell. 2021 Feb 18;184(4):983-999.e24. doi: 10.1016/j.cell.2021.01.018. PMID: 33606986; PMCID: PMC7899134.
3. Glassman CR, Mathiharan YK, Jude KM, Su L, Panova O, Lupardus PJ, Spangler JB, Ely LK, Thomas C, Skiniotis G, Garcia KC. Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells. Cell. 2021 Feb 18;184(4):983-999.e24. doi: 10.1016/j.cell.2021.01.018. PMID: 33606986; PMCID: PMC7899134.
4. Floss DM, Schröder J, Franke M, Scheller J. Insights into IL-23 biology: From structure to function. Cytokine Growth Factor Rev. 2015 Oct;26(5):569-78. doi: 10.1016/j.cytogfr.2015.07.005. Epub 2015 Jul 11. PMID: 26195433.