KC-6448

293T-STAT4-Luc2-IL12RB1-IL12RB2 Cell Line

65453

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Background of 293T-STAT4-Luc2-IL12RB1-IL12RB2 Cell Line

IL-12 binds to the heterodimeric receptor IL12RB1/IL12RB2 and activates downstream STAT4, initiating Th1 immune responses and anti-tumor immunity. Parental 293T cells lack endogenous expression of these receptor subunits and STAT4, making them unsuitable for direct IL-12 pathway activity detection. We generated the engineered cell line 293T-STAT4-Luc2-IL12RB1-IL12RB2 with stable overexpression of IL12RB1, IL12RB2 and STAT4, alongside a Luc2 luciferase reporter responsive to STAT4 activation. This cell model allows quantitative luminescence measurement of IL-12 signaling intensity, suitable for rapid screening of IL-12 modulators and functional validation of the IL-12/STAT4 axis in tumor immunology research.

Specifications

Catalog Number	KC-6448
Cell Line Name	293T-STAT4-Luc2-IL12RB1-IL12RB2 Cell Line
Clone Number	4C3
Host Cell Line	293T-STAT4-Luc2
Description	Stable 293T-STAT4-Luc2 cell line expressing exogenous IL12RB1 and IL12RB2 gene
Quantity	One Vial 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% basal medium+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+75µg/mL hygromycin B+0.5µg/mL puromycin
Selection Marker	hygromycin B, puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-STAT4-Luc2-IL12RB1-IL12RB2 cell line was generated using a lentiviral vector expressing IL12RB1 and IL12RB2 sequence.

Characterization

Figure 1. 293T-STAT4-Luc2-IL12RB1-IL12RB2 cell line was seeded into the 96-well plate, and treated with IL-12 at a maximum concentration of 1000ng/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the folHighing day.

References

Thierfelder W E, van Deursen J M, Yamamoto K, et al. Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells [J]. Nature, 1996, 382 (6587):171-174.
Presky D H, Yang H, Minetti L J, et al. A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits [J]. Proc Natl Acad Sci U S A, 1996, 93 (24):14002-14007.
Trinchieri G. Immunology of IL-12: An update on functional activities and implications for disease [J]. Front Immunol, 2020, 11:619008.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.