Two cell lines are co-cultured in a 96-well plate, cell line A is eGFP-tagged and B is not. To see whether A line is affected by B (after specific treatments), I need to check eGFP signals in a plate reader. I still need to transduce the cell line with an eGFP-harboring lentivirus. I know that luciferase-transduced cell line has already been applied to relevant experiments but I have to apply eGFP system and curious whether it works in a fluorescence plate reader.
Any experience or thought in assays alike is appreciated.
If your cells start expessing eGFP you will measure fluorescence directly in plate.
Be sure about wells with appropriate controls since plastik and media properties could significantly affect fluorescence spectra.
Yes, you can certainly read EGFP on plate reader. Although it depends on your EGFP expression levels.
I was measuring a low EGFP expressing cell line to go through high through put platform. You can use FLUOstar, or Polarstar from BMG Labtech you can also use Envision or Victor from Pelkin Elmer or other related plate reader. Keep in mind that if your EGFP expression is low, you need consider to use optical plates.
Thank you guys, I did not know that eGFP signals are also measurable through a plate reader. Lingli, I was also thinking that specific readers should be used (not any one), I mean with sett-up systems like FACS, right?, tomorrow I will check with FACS whether eGFP is already there and get back to you.
In order to get the best signal and lowest noise from measurements in living cells you should measure from the bottom of the well. This brings the optic group closer to the cell layer and gets rid of the negative influence of the culture medium. Moreover, a focusing system is a huge advantage as it can focus your light beam directly on the cell layer. For bottom reads though you need, in case of fluorescent measurements, black plates with clear (transparent) bottom.
Finally a plate Reader that is able to scan the bottom of the well is advantageous. Some times cells unevenly distribute on the bottom of the well and if you measureonly in the Center of it you may lose a lot of signal simply because only few cells are there.
Different Readers, including BMG LABTECH, offer the possibility to scan the well with different resolutions.
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