Can anyone provide a protocol or standard for choosing the number of cells to analyze for live cell FRET investigations? I have been trying to be as unbiased as possible in my selection of cells, so I typically just analyze every cell in the field of view, but that can sometimes be upwards of 40-50 cells, so I am wondering if there is a more reproducible way of analyzing. Thanks in advance.
I draw a Region of interest around all the cells in my field of view and see if the cells respond.
for analysis, then I will select only those fret traces which actually show a fret trace over the background and after doing all the fret corrections.
Amod Godbole When you say they "show a trace over the background", do you draw an ROI in an area of the background to get that value?
Tyler Johnson yes, I draw a ROI over a background which is devoid of any cells
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