Overview
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.
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Materials
Name | Company | Catalog Number | Comments |
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 |