6 replies to this topic

[Grenouille]

Hi everyone!

I started to do a lot of FACS analysis (which is something I never did before) of viral GFP transduced cells and did my compensations by using single stained cells. I only get a small GFP signal and would like to use compensation beads to do my compensations but really don't know whether it is possible to do my GFP compensations by the use of FITC stained beads.
This might be a stupid question, but I am happy about every answer.

Thank you all!

[leelee]

In my opinion, no you can't.

Have you tried emailing the BD tech experts to see what they recommend?

[zodiac1505]

I think maybe you can. Since GFP comes up in the FITC channel, it might work.

[leelee]

Ok so I've thought more about it and no you definitely cannot.

GFP and FITC are unique flurochromes, so they will have their own unique spillover into the other channels. So a compensation with one for the other wouldn't be useful.

If you head over to the spectra viewer here, and enter GFP and FITC, you'll see what I mean.

[zodiac1505]

Yeah, leelee is right. The compensations won't be accurate.

[Grenouille]

Thank you all. That's what I thought too. So I will continue to do my compensations with gfp positive cells what sometimes makes my experiments much more difficult (e.g. because of the low gfp intensities).
I also searched for something like gfp labeled beads (if something like this exists) but so far found nothing.

Again thank you all,

Grenouille

[zodiac1505]

Are you making viruses and if you are, are you sure your transfections and viral production are going fine? Maybe you might want to try out different reagents to get better transfection efficiencies.