A Lentiviral Transcriptional Reporter System to Generate a Glioblastoma Stem Cell Differentiation Reporter Line
Transcript
To begin seed 1 million glioblastoma stem cells or GSCs in 2 milliliters of complete neural stem cell growth medium in a 6-well plate. Next, transduce the cells with the lentivirus reporter at a multiplicity of infection equal to five.
To the cell and virus mixture, add 2 microliters of polybrene such that the final concentration is 8 micrograms per milliliter, and incubate the cells at 37 degrees Celsius and 5% CO2 overnight. After incubation, replace the growth medium to remove unbound virus, and centrifuge cells at 360 times g for 5 minutes. Then, carefully aspirate medium and plate cells back in a 6-well plate, and continue to incubate the cells for 24 hours.
Harvest 0.5 milliliters of the total culture volume. Pellet cells by centrifugation at 360 times g for 5 minutes at room temperature. Resuspend cells in 0.5 milliliters fresh neural stem cell growth medium.
Place the plate back in the incubator. To allow for expansion for at least 72 hours. Add 200 microliters of dissociation reagent, and incubate for five minutes in a water bath set to 37 degrees Celsius. Dissociate cells by pipetting up and down gently. To the dissociated cells, add 800 microliters of HBSS to ensure minimal cell attachment or aggregation.
Next, transfer 200 microliters of each cell suspension into wells of a 96 multi-well plate. Determine the percentage of cells expressing the green fluorescent protein by flow cytometry. Record at least 10,000 viable cells in each acquisition.
