a question about screening transfected cells by GFP protein - (Feb/07/2007 )

I would like to know which equipment is better to detect GFP reporter protein between FACS and Fluorescence microscorpy. Recently I have no more cells to do both things. I would like to choose one to save my time. please give me any suggestion.
ppapon

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I would go with FACS. more sensitive.

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give both a go at leats you will have some pretty pictures!!

though i would go for microscopy i have been using facs to detect the transfection efficiency of a gfp containing plasmid into my cells and found the peak shift to be only very slight

upon microcopical anlaysis pretty much all of my cells were transfected.

could just be my facs machine !!!

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it depends on what you like to show: more quantitative (FACS) or more (sub)cellular specific imaging of single cells; as microscopy is also to use for quantification of signals you may decide to go ahead with fluorescence microscopy

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Dear ppapon,

I see no reason why you can't do both. Normally you can look at your cells under the fluorescence microscope while they are still attached to your plates/flasks. Unless you mean confocal microscopy in which case you'd have to grow them on coverslides anyway. GFP is stable enough that you won't bleach it out by looking at them (unless you take hours and hours).

After you had a quick look then you can harvest the cells and do FACS to get an accurate percentage of green cells. I always check my cells under the microscope before I do FACS just to make sure it's worth doing the FACS.

It is not true that FACS is more sensitive, it's just more accurate when it comes to numbers. Usually when you have GFP expression you can see it under the fluorescence microscope with the bare eye. If you have to use long exposure times (>2 sec on your CCD camera) then it might in fact be autofluorescence.

LeserattePD

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Dear Jimmy,

For proper FACS setup you should always have a negative control (ie cells of the same type that are mock-transfected and therefore not green). This is your baseline to which you can compare your positive cells (under the same machine settings).

If you did this but did not see a shift in the FACS I would be very careful about what exactly you think you are seeing under the microscope. As I explained above it is quite easy to mistake autofluorescence for GFP positive cells if your exposure times are high.
In my experience GFP expresses very well from the Clontech original plasmid that has a CMV promoter and if you can see it with your eyes under the microscope it should be at least a one log shift on the FACS. Other promoters/IRES sequences might be less eficient.

However FACS machines and the correct instrument settings are a very complex thing and if you could see your green cells under the microscope with less than 1 sec exposure but not on the FACS (compared to the neg control sample) then I'd suspect your FACS settings were wrong.


LeserattePD

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