KC-4335

293T-GRE-Luc2-IL4Ra-IL2RG Cell Line

54375

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Background of 293T-GRE-Luc2-IL4Ra-IL2RG Cell Line

Glucocorticoids (GCs) and their analogs regulate downstream gene expression through the glucocorticoid receptor (GR, also known as NR3C1), which is a member of the nuclear receptor superfamily of ligand-activated transcription factors. GCs diffuse into the cytoplasm, bind to GR, and are then translocated to the nucleus, where they bind to glucocorticoid response elements (GREs) in the promoters of target genes, thereby activating or repressing gene expression. GCs also modulate gene expression via non-GR pathways, such as through cAMP response element-binding (CREB) protein, activator protein (AP)-1, and NF-κB. These receptors have several major domains, including an amino-terminal activation domain, a central zinc-finger DNA-binding domain, and a carboxy-terminal ligand-binding domain. This system can be used to study the activation levels of glucocorticoid-mediated signaling pathways, drug research, and gene overexpression and RNAi phenotypic analysis. The primary interleukin-4 (IL-4) receptor complex on monocytes (type I IL-4 receptor) includes the 140-kDa alpha chain (IL-4R alpha) and the IL-2 receptor gamma chain, gamma(c), which heterodimerize for intracellular signaling, resulting in suppression of lipopolysaccharide (LPS)-inducible inflammatory mediator production.

Specifications

Catalog Number	KC-4335
Cell Line Name	293T-GRE-Luc2-IL4Ra-IL2RG Cell Line
Clone Number	14#
Host Cell Line	293T-GRE-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of IL4Ra-IL2RG signaling pathway
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 +150μg/mL Hygromycin+1μg/mL Puromycin
Selection Marker	Puromycin, Hygromycin B
Morphology	Epithelial
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

293T-GRE-Luc2-IL4Ra-IL2RG cell line was generated using lentivirus expressing IL4Ra-IL2RG sequence.

Characterization

Figure 1: Different passage times of the 293T-GRE-Luc2-IL4Ra-IL2RG cell line was seeded into the 96-well plate, and treated with ADC and control at a maximum concentration of 20μg/mL for 16 hours, then readout with Bright-Glo system.

Figure2: 293T-GRE-Luc2-IL4Ra-IL2RG cells were seeded into 96-well plates, treated with Dexamethasone for 24 hours, and then read out using Bright-Glo Detection System.

Figure 3: Characterization of IL4R overexpression in 293T-GRE-Luc2-IL4Ra-IL2RG stable clones using FACS.

Figure 4: Characterization of IL4R overexpression in 293T-GRE-Luc2-IL4Ra-IL2RGstable clones 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 1:4-1:6 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

Bankaitis KV, Fingleton B. Targeting IL4/IL4R for the treatment of epithelial cancer metastasis. Clin Exp Metastasis. 2015 Dec;32(8):847-56. doi: 10.1007/s10585-015-9747-9. Epub 2015 Sep 18. PMID: 26385103; PMCID: PMC4651701.
Liu B, Zhang TN, Knight JK, Goodwin JE. The Glucocorticoid Receptor in Cardiovascular Health and Disease. Cells. 2019 Oct 9;8(10):1227. doi: 10.3390/cells8101227. PMID: 31601045; PMCID: PMC6829609.
Wu T, Shao Y, Li X, Wu T, Yu L, Liang J, Zhang Y, Wang J, Sun T, Zhu Y, Chang X, Wang S, Chen F, Han X. NR3C1/Glucocorticoid receptor activation promotes pancreatic β-cell autophagy overload in response to glucolipotoxicity. Autophagy. 2023 Sep;19(9):2538-2557. doi: 10.1080/15548627.2023.2200625. Epub 2023 Apr 20. PMID: 37039556; PMCID: PMC10392762.
Oakley RH, Cidlowski JA. The biology of the glucocorticoid receptor: new signaling mechanisms in health and disease. J Allergy Clin Immunol. 2013 Nov;132(5):1033-44. doi: 10.1016/j.jaci.2013.09.007. Epub 2013 Sep 29. PMID: 24084075; PMCID: PMC4084612.
Hart PH, Bonder CS, Balogh J, Dickensheets HL, Donnelly RP, Finlay-Jones JJ. Differential responses of human monocytes and macrophages to IL-4 and IL-13. J Leukoc Biol. 1999 Oct;66(4):575-8. PMID: 10534111.