A Lentiviral Transcriptional Reporter System to Generate a Glioblastoma Stem Cell Differentiation Reporter Line

1. Lentiviral Transcriptional Reporter System

NOTE: In all flow cytometric analyses, use parental, non-transduced, cells or vector-transduced (non-fluorescent) cells to establish baseline fluorescence. Also, note that in all steps where mechanical trituration is called for, be gentle. Harsh trituration can kill a significant number of the fragile GSCs and influence flow cytometry results.

1. Plate 1×10⁶ cells in 2 mL of complete neural stem cell growth medium in a 6-multiwell plate.
2. Transduce cells by adding lentivirus reporter at a multiplicity of infection (moi) equal to 5.
3. Add 2 µL of polybrene for a final concentration of 8 µg/mL.
4. Incubate the cells at 37 °C and 5% CO₂ overnight.
5. The next day, replace growth medium to remove unbound virus. Place the plate back in the incubator at 37 °C and 5% CO₂ and incubate for 24 h.
6. Harvest 0.5 mL of the total volume; spin down cells at 360 x g for 5 min at room temperature in a 15- mL conical tube and remove supernatant by aspiration. Add 0.5 mL of fresh neural stem cell growth medium to the remaining cells and return the plate to the incubator for continued expansion.
7. Add 200 µL of dissociation reagent and incubate for 5 min at 37 °C.
8. Dissociate cells by gently pipetting up and down.
   NOTE: Harsh trituration may result in significant killing of GSCs; complete dissociation is usually achieved after 20 – 30 times.
9. To minimize cell attachment or aggregation, add 800 µL of Hank's Balanced Salt Solution (HBSS) for a final volume of 1 mL.
10. Transfer 200 µL of each cell suspension to a well of a 96-multiwell plate.
11. For this procedure, use a benchtop flow cytometer with 96-well plate capability equipped with a blue laser for excitation and with the capability of detecting green fluorescent protein. Perform flow cytometric analysis with at least 10,000 viable cells for each acquisition.
12. Determine the percentage of GFP-positive cells by flow cytometry.

2. Determination of GFAP:GFP Expression by Flow Cytometry

1. Harvest an aliquot of cells (0.5 mL) from each reporter clone and non-transduced controls to determine the exact percentage of GFP-positive cells. Considering reporter clones that contain ~1 – 5% GFP-positive cells.
2. Spin cells at 360 x g for 5 min at room temperature and remove supernatant by aspiration.
3. To each pellet, add 200 µL of cell dissociation reagent and incubate the tubes in a water bath set to 37 °C.
4. Triturate cells gently to achieve a single-cell suspension (usually between 20 to 30 times).
5. To minimize cell attachment or aggregation, add 800 µL HBSS to a final volume of 1 mL.
6. Transfer 200 µL per well of a 96-multiwell plate from each cell suspension.
7. For this procedure, use a benchtop flow cytometer with 96-well plate capability. Perform flow cytometric analysis with at least 10,000 viable cells for each acquisition.