This article discusses the preparation of glioma stem cells (GSCs) tagged with luciferase for high throughput imaging assays in drug screening. The process involves culturing GSCs, tagging them with a virus containing luciferase-enhanced green fluorescent protein, and visualizing the results under a fluorescence microscope.
Glioma stem cells or GSCs are a subpopulation of glioma - a cancer of glial cells in the brain.
Tagging these GSCs with bioluminescent enzymes like luciferase helps establish high throughput imaging assays for drug screening.
To prepare luciferase tagged GSCs, begin with a culture of GSCs in a suitable growth medium.
Centrifuge the culture to obtain a cell pellet and remove the supernatant.
Add suitable digestive enzymes to the pellet. These enzymes dissociate cell clusters and degrade any traces of connective tissue to obtain a single-cell suspension.
Now, centrifuge the single-cell suspension and remove the supernatant. Resuspend the pellet in a fresh medium.
Next, seed the desired concentration of cells in a culture plate.
Incubate the culture and allow the cells to adhere to the culture plate.
Subsequently, add the desired titer of a virus-containing-gene construct for luciferase-enhanced green fluorescent protein or EGFPand incubate.
The virus interacts with the surface receptors of GSCs, triggering the release of viral RNA containing luciferase-EGFP. Cellular enzymes catalyze reverse transcription of RNA to double-stranded DNA and integrate it into the genome of GSCs.
When visualized under a fluorescence microscope, luciferase-expressing cells appear green due to the fluorescence emitted by EGFP.
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.
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