Encyclopedia of Experiments: Cancer Research
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Luciferase-tagged Glioma Stem Cell Model - ConceptTranscript
Begin by collecting the glioma stem cells or GSCs from the culture medium and centrifuging them at 70 x g for 3 minutes at room temperature. After removing the supernatant, digest the cells with accutase for 4 minutes at 37 degrees Celsius.
Using a 200-microliter tip, pipette the cells repeatedly to dissociate and resuspend the cell pellet. Add the GSCs at a density of 200,000 cells in 1 milliliter of culture medium into each well of a 12-well culture plate and incubate them overnight.
On the next day, add 30 microliters of luciferase-EGFP virus supernatant into each well of the plate. Centrifuge the cells at 1,000 x g for 2 hours at 25 degrees Celsius and incubate them overnight.
On the following day, replace the medium in the wells by collecting the GSCs, centrifuging them, removing the supernatant, resuspending them in fresh medium, and replating them. Culture the cells for another 48 hours.
Observe the plate under a fluorescent microscope and confirm the appearance of GFP-positive cells.
