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

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

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.

Materials

List of materials used in this article

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