Hi fellow researchers!
I would like to know your thoughts on the detailed steps to use cells transfected with GFP-tagged plasmid for subsequent drug studies, etc.
I currently have transfected SHSY5Y cells in 12 well plates with plasmid encompassing A53T (for mutant alpha synuclein) tagged to GFP. After 24 hours, I was able to observe bright green fluorescence under the microscope.
I used lipofectamine 2000 and there are differing views on its potential to achieve stable transfection. I have been advised that it is indeed possible to achieve stable transfection if the cells are trypsinized and plated again with selective media (keep for 2-4 weeks before colony selection). I also wonder if I could take my current cells and give them for FACS sorting? Would that be a better and faster way to choose the cells expressing my protein?
My ultimate goal is to use these cells to test my nanoparticle's efficacy on reducing a-syn aggregation. While some studies have simply used the transiently transfected cells for their studies, some go on to make stable lines before using them for other studies.
What should be my workplan right now? I am considering first checking the expression levels of my cells with qPCR and western blotting. If I were to use my current expressed cells (probably transient), is it ok if I passage and replace them in 6 well plates, and directly do my nanoparticle studies from there for subsequent analysis with imaging, qPCR and western blotting?
Note: I do not necessarily require stable lines, but I am confused as to how I should proceed if I decide to just use my transiently transfected cells.
Thank you!
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