I transduced my B cells with GFP containing plasmids using lentivirus and achieved 60% positivity. However, when I checked my cells under the confocal microscope, I can not see any GFP signals. (Maybe a little bit, but the signal it gave is similar to the signal given by non-transduced B cells). I was wondering does anyone know what could happened?
Possibility-1: Autofluorescence detected in FACS (You need a set of untransfected controls to exclude autofluorescence).
Possibility-2: You may use a wrong filter in microscope.
I would always trust the eyes (look through the eye piece in microscope to see if you can see GFP).
Good luck.
Serena Wan I think transfection was inefficient. Considering that you are using Blue excitation (ex. 420 nm) very few or no plasmid has been transfected. Whatever you are seeing in the confocal microscope, it's the auto-fluorescence as confocal microscopy is well known to enhance very weak signals. Whatever you are getting in FACS, compare it with a negative control i.e. run FACS with B cells having no plasmid transfected. I think you will get the same MCF.
All the best.
Flow cytometer is much more sensitive than confocal microscope. I have several experiemce when a weak GFP-expressing cell is easily detectable by flow cytometer but direct imaging cannot see anything. Try increase your microscope laser intensity may help a little, but background will increase too.
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