Transfection, GFP, FACS and UV Microscope - great inconsistency b/t FACS and UV microscope (Aug/25/2005 )

Hi all. I recently transfected a HEK293-derived cell line with pZsGreen1-N1 (Clontech). I wanted to monitor the transfection efficiency so I:

1) looked at the cells (plated in TC dishes) under a UV microscope --> confluency was high but one can clearly see that <50% of the cells were green and the rest all looked identical to the untransfected control;

2) ran the cells through a FACSCalibur --> nearly 100% of the cells were positive for fluorescence whereas the control was what you would expect -- all negative.

I tend to want to trust the UV microscope as it is simple and can be seen with my own eyes. However, I'd just like to know why there might be such a gigantic discrepany between the results derived from the two methods. Any input or feedback would be greatly appreciated.

Hi, Omegaxx!


The only thing that could happen that you indeed had nearly 100% transfection efficiency. FACSCalibur is more sensitive then human eyes.

good luck,

A.

Maybe wait a little longer before you read with microscope, it might take some time for all the cells to produce enough protein to be visible with the human eye.

FACS is definitely more sensitive. The eye really doesn't see faint color very well at all and besides that your microscope bulb could be old, the filters not 100% correct, the lenses dirty etc etc etc. If in doubt of autofluorescence or something you should run a negative/non-transfected/mock transfected line as a control.