Transfection of fluorescent chimeras should be performed as appropriate for the chosen cell type. Typically, cells are plated on 25-mm circular #1 thickness cover glasses, placed within a well of a six-well plate, transfected with a cationic transfection reagent, or electroporated and then plated. Cells are typically imaged 24 h after transfection. The following samples should be prepared.
YFP-fusion alone CFP-fusion alone CFP-fusion + YFP (free) YFP-fusion + CFP (free) CFP-fusion + YFP-fusion
Control for spectral cross talk Control for spectral cross talk Negative control for FRET Negative control for FRET Experimental sample
Spectral cross talk is unique to each microscope configuration and should be measured for each fluorophore. It should be monitored frequently and recalculated as described later whenever changes to the microscope optics are made. In addition to the aforementioned samples, it is also extremely beneficial to express a set of CFP/YFP fusion proteins known to interact and provide ample energy transfer to use as a positive control.
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