Can I use fluorescence microscopy to assess shRNA plasmid transfection efficiency in my model cell line of interest, when using a vector expressing a GFP reporter gene?

Whenever possible, we recommend using flow cytometry to determine the transfection efficiency of plasmids which express GFP. If you must use fluorescence microscopy, we recommend staining the cell culture with a fluorescent nuclear (DNA) stain, such as one of the Hoechst dyes, so you can count both the number of transfected cells and the total number of cells while looking at the same fluorescent view of the cells. Also, count the cells in a number of randomly chosen but representative fields and not just one field. We do not recommend comparing the phase (cells) and fluorescence (GFP) views of the cells to subjectively estimate the percentage of transfected cells, as this method tends to overestimate efficiency.

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