Luciferase-tagged Glioma Stem Cell Model: A Lentivirus-mediated Method to Prepare Luciferase-tagged Glioma Stem Cells for In Vitro Therapeutic Studies

Samenvatting

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

1. Begin with the GSCs culture and centrifuge at 70 x g for 3 min at room temperature.
2. 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.
3. Dilute the cells to 2 x 10⁵ cells per well in a 12-well culture plate and culture the cells overnight.
4. 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.
5. Refresh the medium the next day and culture the cells for another 48 h.
6. Observe the cells under a fluorescent microscope to confirm the appearance of the GFP-positive cells.
7. Use a flow cell sorter to sort and select the GSCs with high GFP fluorescence to culture the cells further.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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