Dear Puneeta

microplate reader is less sensitive than microscopy a the detection depends not only from the trasfection efficiency but also from the intensity of each cells and therefore is it possible that some optimization are required also because the signal is affected from autoflorescence, scattering.

just some questions (maybe stupind) to understand where can be your issue:

1) Are you expressing the wt GFP or EGFP sequence? Because with the Wt GFP is it preferable performing excitation at 395 nm (https://www.biotek.com/resources/technical-notes/excitation-and-emission-of-green-fluorescent-proteins/)

2) At which wawelenght did you acquire the emission?

3) Are you using black or transparent plates to detect the fluorescence ?

4)Are the cells are growing in suspension or in adesion at the bottom of the plates?

5) Which is your cell density at the moment of measurment?

6) no differences beetween untrasfected and trasfected means low signal for both or high signal for both but no increase in the trasfected?

Ciao

Manuele

What type of microplate are you using? This might help?

https://www.bmglabtech.com/which-is-the-best-microplate-for-my-assay/

Although we tend to detect GFP using an ELISA, rather then by direct fluorescence

I think it depends on the microplate you used. a black microplate may be useful.

Also, 1) what is the emission wave length ?

2) try to increase the band width from 5 to 7 or 10.