transfection of cells with two fluorescent reporters - (Feb/07/2015 )

Hi everyone,

I have a question about transfection and imaging fluorescence. I have two cell lines - one transfected with GFP and one transfected with mcherry - and I would like to transfect them both with another fluorescent marker (ex: YFP) so when estrogen receptor element is turned on, you can see YFP. Some background here: the reason why I need my two cell lines transfected with GFP and mcherry is because I will be coculturing them and need to tell them apart. I realize that it is much more common for luciferase to be used as a marker for ERE, however, I will be using a confocal microscope for imaging so luciferase is not an option. My question is: can I transfect a fluorescent marker in a cell with another fluorescent marker? How will this affect imaging? 

There is no reason why you can't transfect more than one marker. Just make sure that the excitation and emission spectra between the markers are sufficiently different that you won't get bleed-through.  You should also make sure that you have the appropriate selection marker for your transfected plasmid - e.g. the GFP cells might have been transfected with a G418 selection so they will be resistant to it already, so you need another selection marker on the transfected plasmid.