Can I use fluorescence microscopy to assess the transfection efficiency of the SureSilencing shRNA Plasmids with GFP in my model cell line of interest?

Whenever possible, use flow cytometry to determine the transfection efficiency of the SureSilencing shRNA Plasmids encoding GFP. We recommend using the standard FACS settings of an argon laser (488nm excitation) and an emission filter of 530+15nm. Transfected cells can be visualized microscopically using standard fluoroisothiocyanate (FITC) filters. We typically use an excitation filter of 470+20nm and an emission filter of 515nm (long pass). When utilizing fluorescence microscopy, we recommend staining the transfected cells with a fluorescent nuclear (DNA) stain, such as one of the Hoechst dyes, to 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. 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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