Luciferase-tagged Glioma Stem Cell Model: A Lentivirus-mediated Method to Prepare Luciferase-tagged Glioma Stem Cells for In Vitro Therapeutic Studies
Published: April 30, 2023
Abstract
Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells. J. Vis. Exp. (2021)
This video demonstrates the establishment of luciferase-tagged glioma stem cells via lentivirus-mediated gene delivery. Luciferase acts as a reporter gene and helps facilitate rapid drug screening experiments.
Protocol
1. Preparing Luciferase-tagged GSCs
- Begin with the GSCs culture and centrifuge at 70 x g for 3 min at room temperature.
- Remove the supernatant and digest the cells with accutase for 4 min at 37 °C. Use a 200 µL tip and pipette repeatedly to dissociate and resuspend the cell pellet.
- Dilute the cells to 2 x 105 cells per well in a 12-well culture plate and culture the cells overnight.
- Add 30 µL luciferase-EGFP virus supernatant (titer >108 TU /mL) into each well in the plate and then centrifuge the cells at 1,000 x g for 2 h at 25 °C. Culture the cells overnight.
- Refresh the medium the next day and culture the cells for another 48 h.
- Observe the cells under a fluorescent microscope to confirm the appearance of the GFP-positive cells.
- Use a flow cell sorter to sort and select the GSCs with high GFP fluorescence to culture the cells further.
Divulgations
The authors have nothing to disclose.
Materials
| B-27 | Gibco | 17504-044 | 50X |
| EGF | Gibco | PHG0313 | 20 ng/ml |
| FGF | Gibco | PHG0263 | 20 ng/ml |
| Gluta Max | Gibco | 35050061 | 100X |
| Neurobasal | Gibco | 21103049 | 1X |
| Penicillin-Streptomycin | HyClone | SV30010 | P: 10,000 units/ml S: 10,000 ug/ml |
| Sodium Pyruvate | Gibco | 2088876 | 100 mM |
Tags
Citer Cet Article
Luciferase-tagged Glioma Stem Cell Model: A Lentivirus-mediated Method to Prepare Luciferase-tagged Glioma Stem Cells for In Vitro Therapeutic Studies. J. Vis. Exp. (Pending Publication), e20613, doi: (2023).