Hey,
I try to transfect primary human dermal fibroblasts. For this, I used several transfection reagents. One of it is DNA-IN (amsbio). A few cells are definitely transfected (strong GFP fluorescence).Others show only quite a weak fluorescence, so I ask myself, if this is just autofluorescence or if these cells are really transfected (see pictures attached).
Thanks in advance,
Basty
If you could analyze the cells on a flow Cytometry then you'd get your answer. Flow is generally more sensitive and easily quantifies eGFP expression relative to a control. The control should be mock transfected, as the transfection procedure can affect auto fluorescence properties.
You can also activate an additional channel in FACS to see the level of autoflourescent. This additional channel will not be the flouresncent that is being expressed in your samples but would be another channel.
From the images you have attached, it seems like the GFP as a huge background. This may be due to your microscopy settings as well. To eliminate this, as suggested by others you try to sort the cells by FACS, provided that the sample size is not an issue. Otherwise, try imaging in a higher magnification to come to a better conclusion.
Some of the weaker fluorescent cells could still be positive.
If you want to be certain analyse them by flow or if thats not an option, seed them on small glass coverslides, fix them and do an antiGFP IF staining with a different fluorochrome and add a DAPI stain. Add proper single stain controls and you should have your answer.
Consider to remove the background, I feel you increase the stimuli of laser too much. DAPI would be a good control to minimise the saturation. eGFP detection has a better chance by FLOW.
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