KC-1455
293T-ISRE-Luc2 Cell Line
Background of 293T-ISRE-Luc2 Cell Line
The ISRE (interferon stimulated response element) reporter gene is mainly used to detect the transcriptional activity of STAT1/STAT2 in the cell Type I Interferon signaling pathway, drug research, gene overexpression and RNAi phenotypic analysis. Interferon (IFN)-I and IFN-II both induce IFN-stimulated gene (ISG) expression through Janus kinase (JAK)-dependent phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT2.
Specifications
| Catalog Number | KC-1455 |
|---|---|
| Cell Line Name | 293T-ISRE-Luc2 Cell Line |
| Host Cell Line | 293T |
| Description | 293T cell line stable expressing exogenous luciferase gene under the control of ISRE responsive element |
| 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% RPMI1640 + 20% FBS + 10% DMSO |
| Propagation Medium | DMEM + 10% FBS +140μg/mL Hygromycin B |
| Selection Marker | Hygromycin B |
| Morphology | Lymphoblast |
| Subculture | Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1 × 105 cells/mL |
| Incubation | 37 °C with 5% CO2 |
| Storage | Liquid nitrogen immediately upon receiving |
| Doubling Time | Approximately 24 hours |
| Mycoplasma Status | Negative |
| In Vivo Validation | NA |
Cell Line Generation
293T cell line was generated using lentivirus vector expressing ISRE and luciferase sequence.
Characterization
Figure: 293t-isre-luc2 cell line was seed into the 96-well plate, and treated with IFN-a2A at a maximum concentration 10000 units/mL diluted 3.16-fold for 16 hours, then readout with Bright-Glo system.
Cell Resuscitation
- Prewarm culture medium (DMEM supplemented with 10% FBS, 140μg/mL Hygromycin B).
- 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 (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
1. Michalska A, Blaszczyk K, Wesoly J, Bluyssen HAR. A Positive Feedback Amplifier Circuit That Regulates Interferon (IFN)-Stimulated Gene Expression and Controls Type I and Type II IFN Responses. Front Immunol. 2018 May 28;9:1135.
2. Uccellini MB, García-Sastre A. ISRE-Reporter Mouse Reveals High Basal and Induced Type I IFN Responses in Inflammatory Monocytes. Cell Rep. 2018 Dec 4;25(10):2784-2796.e3.