KC-0873
293T-IL4R-Cell-Line
Background of 293T-IL4R-Cell-Line
IL4 receptor complex is composed of IL-4Ra subunit and one accessory protein, the common cytokine receptor chain or IL-13Ra1, both of which transduce signals through IL4Ra. Type I IL-4R complexes, composed of IL-4Ra and common cytokine receptor, dominate in hematopoietic lineages and exclusively bind to IL-4 but not to IL-13. Type II IL-4R complexes are composed of IL-4Ra and IL-13Ra1. They are present on both hematopoietic and nonhematopoietic tissues and bind to both IL-4 and IL-13.
Specifications
| Catalog Number | KC-0873 |
|---|---|
| Cell Line Name | 293T-IL4R-Cell-Line |
| Host Cell Line | Human HEK293T cell line |
| Description | Stable HEK293T cell line expressing exogenous human IL4Ra & IL13R1 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 1:3-1:6 every 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 human type II IL4R cell line was generated using a plasmid vector expressing human IL4Ra and IL13R1sequence.
Characterization
Figure: Characterization of IL4R overexpressing in the 293T stable clone using FACS.
Cell Resuscitation
- Prewarm culture medium (DMEM + 10% FBS + 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 1:3-1:6 every 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
- Kashiwada, M., Giallourakis, C. C., Pan, P.-Y., & Rothman, P. B. (2001). Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation. The Journal of Immunology, 167(11), 6382-6387. https://doi.org/10.4049/jimmunol.167.11.6382
- Ikizawa, Koichi, and Yukiyoshi Yanagihara. 2000. ¡ùPossible Involvement of Shc in IL-4-Induced Germline ÈÖ Transcription in a Human B Cell Line.¡ì Biochemical and Biophysical Research Communications 268 (1): 54ÿ59. doi:10.1006/bbrc.2000.2080.
- Maes, T, G F Joos, GG Brusselle American journal of respiratory cell, 2012. n.d. ¡ùTargeting Interleukin-4 in Asthma: Lost in Translation.¡ì Atsjournals.org