Indirect_FRET

Indirect FRET analysis in primary cells

Objectives:

Explore the proximity between two molecules by probing them with AF488 (donor) conjugate and AF546 conjugate (acceptor) antibodies. This proximity is evaluated by energy transfer between donor and acceptor probes. The energy transfer or FRET efficiency is proportional to the proximity of these two probes.

Disadvantages: The limitation to this method concerns the quantification of this proximity. Indeed, since this is an indirect staining implicated two or more antibodies, we can only evaluate the difference on a semiquantitative manner (lower or higher).

Advantages: Use of physiologic and non-modified cells. Contrary to conventional FRET, this method does not require any transfection or genetic modification. Consequently, results coming this analysis are more physiologically relevant.

Step-by-step tutorial

1. download the script bastien_Fret.ijm
   Right click on RaW > Save As... (please save as .ijm)
2. download imageJ/Fiji
3. Update ImageJ/Fiji > Help > Update...
4. Restart ImageJ
5. Drag and drop the script into imageJ/Fiji and run it