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I transfected HEK293t cells with pLeGO vector containing GFP to investigate the calcium phosphate method. When examined with a fluorescent microscope, the brightness of my cells under the microscope is very low and almost looks like autofluorescence, but when examined by FACS, the expression level of GFP+ cells was reported to be about 78% compared to the control group (0.5%). My question is, which of these methods can I trust? Eye observation method with fluorescent microscope or flow cytometry method?
I would certainly trust the cytometry data on this. The cytometer's detector are way more precise and accurate than your eyes1
Although microscopy and flow cytometry employ similar approaches, comparing signal strength between the two can be challenging. Microscopy relies on the sensitivity of the microscope's light source and optic path, which may not always be as sensitive as flow cytometry's light detection system. However, suppose your flow cytometry results show a distinct population not present in the control cells that are properly transfected with a scrambled vector. In that case, you can consider the experiment successful and conclude your work.
If you wish to incorporate microscopy results, you can enhance the GFP signal by using a GFP-specific antibody and utilizing chromophores with 545 nm or 647 nm wavelengths. This approach allows you to separate the GFP-antibody signal from the GFP original 488 signals to obtain clearer results.
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