If you're talking about compensation so that you can measure eGFP without the interference of mCherry signal, then this needs to be set up with the proper controls when running your samples on the flow cytometer.

If you're talking about normalizing your eGFP signal to the mCherry signal, you can use flowjo to calculate the gMFI (geometric mean fluorescence intensity) for eGFP and mCherry. Then you can export the data as a table, and simply divide the gMFI of eGFP by the gMFI of mCherry using Excel or whatever software you prefer. Note that you should only use the gMFI metric if the fluorescent signal is normally distributed across your population of cells.

Hey, so in FlowJo, if you wanna subtract the background mCherry signal from all your cells and basically see GFP relative to that, the easiest way is to make a ratio parameter. Just go to the “Derived Parameters” option in the workspace, create a new one, and set it as GFP divided by mCherry (like FITC-A / PE-Texas Red-A, or whatever your actual channels are). That way, FlowJo gives you a new parameter that tells you how much green signal you’re getting relative to red in each cell. You can then plot that and it automatically accounts for the background. Super handy. Also, make sure your axes are set to logicle scale so things don’t look weird near zero. That’s pretty much it!

thank you so much for all your help, this has really been useful.

I was wondering if you know a way to make some statistics on this ratio parameter? i have multiple different treatments that i would like to compare to see if one strain binds more cyclic di-GMP than another strain, to compare the two strains i would like to calculate either the mean or ideally calculate the area underneat the curve of each histogram over the ratio parameter to compare between treatments. Is there a way to do this?