KC-4034

293T-STAT5-Luc2-TSLPR-IL7Ra-Cell-Line

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Background of 293T-STAT5-Luc2-TSLPR-IL7Ra-Cell-Line

TSLP, is a originally isolated from the mouse thymic stromal cell line, is a pleiotropic cytokine that is a distant parallel to IL-7 and belongs to the type I IL-2 family of short-chain four- α helical bundles. TSLP is mainly expressed in activated lung and intestinal epithelial cells, keratinocytes and fibroblasts, and dendritic cells, mast cells as well as other immune cells may also secrete TSLP. The TSLP conducts signals through a heterologous receptor (TSLPR and IL-7Ra), stimulation induces the activation and phosphorylation of STAT5 (P-STAT5), and the activation of other pathways. In addition,The TSLP is activated by JAK1 (via IL-7Ra) and by JAK2 (via TSLPR). Subsequently, JAK1 and JAK2 activate STAT5A and STAT5B, ultimately prompting the production of IL-4, IL-5, IL-9, and IL-13 and their pro-inflammatory effects.

Specifications

Catalog Number	KC-4034
Cell Line Name	293T-STAT5-Luc2-TSLPR-IL7Ra-Cell-Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of TSLPR-IL7Ra signaling pathway
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Cell model for monitoring TSLPR-IL7Ra signaling pathway
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +150μg/ml Hygromycin+1μg/ml Puromycin
Selection Marker	Hygromycin and 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
In Vivo Validation	NA

Cell Line Generation

293T-STAT5-Luc2-TSLPR-IL7Ra cell line was generated using lentivirus expressing TSLPR-IL7Ra sequence.

Characterization

Figure 1. Characterization of TSLPR-IL7Ra over-expression in 293T-STAT5-Luc2-TSLPR-IL7Ra stable clones using FACS.

Figure 2. 293T-STAT5-Luc2-TSLPR-IL7Ra cell line was seeded into the 96-well plate, and treated with TSLP at a maximum concentration of 10μg/mL for 16 hours, then readout with Bright-Glo system.

Figure 3. 293T-STAT5-Luc2-TSLPR-IL7Ra cell line was seeded into the 96-well plate, then treated with TSLP (100ng/mL), incubated for 16 hours and readout with Bright-Glo system.

Figure 4. Characterization of 293T-STAT5-Luc2-TSLPR-IL7Ra cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/ml Hygromycin and 1μg/ml Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

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

References

Duchesne M,Okoye I,Lacy P.Epithelial cell alarmin cytokines:Frontline mediators of the asthma inflammatory response.Front Immunol.2022 Oct 14;13:975914.doi: 10.3389/fimmu.2022.975914.
Liu YJ.TSLP in epithelial cell and dendritic cell cross talk.Adv Immunol.2009;101:1-25.doi: 10.1016/S0065-2776(08)01001-8.
Duchesne M,Okoye I,Lacy P.Epithelial cell alarmin cytokines:Frontline mediators of the asthma inflammatory response.Front Immunol.2022 Oct 14;13:975914.doi: 10.3389/fimmu.2022.975914.