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Cell Cycle Analysis - DNA+Antigen/GFP
In many cases a reporter molecule is used to assess transfection. This can be a surface marker such as CD8 or it can be GFP (Green Fluorescent Protein). If using FITC-labelled antibodies, cells may be fixed in ethanol and a standard PI protocol may be used.
In the example below cells have been transfected with both CD8 and a viral cyclin. We can identify the successfully transfected cells by their CD8 expression and then look at the DNA profile of these cells. Panel A shows cells transfected with CD8 alone - the DNA profile of these cells is shown in Panel B. Panel C shows cells transfected with CD8 and cyclin, while Panel D shows that the DNA profile of these cells shows the influence of the cyclin on the cell cycle.
However, GFP is a different matter as the ethanol fixation generally required for good DNA CVs will abolish GFP fluorescence as the GFP fluorophore is very sensitive to conformational changes of the protein. There are two ways round this: firstly to use a farnesylated form of GFP which means that the protein localises within membranes in the cell and is largely resistant to the effects of alcohol fixation. Secondly we can use a dye that stains the DNA of live cells in a quantitative manner. There are two capable of this - Hoechst 33342 and DRAQ5. Below shows cells transfected with GFP and stained with Hoechst 33342. Panel A allows us to specifically gate on the GFP population and Panel B shows the DNA profile of the green cells.
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