GFP and PE - (Mar/01/2011 )
I work on GFP positive cells out from a transgenic mouse, expressing GFP in a certain celltype.
If I do flow-cytometry analysis with these cells I canīt see GFP positive cells in combination with PE.
APC seems to be the better combination - but then I just have one colour left (if working on FACSCAlibur).
Does anybody has experiences staining GFP+ cells with PE or PerCP or other colours (that can be read with the 488nm or 635 nm laser)
Thank you very much !!!
Do you compensate your sample? Usually you can detect GFP and PE together, also for GFP weak cells. Oh well, can you compensate on a Calibur?
If you use ToPro3 for DNA (APC channel), works very well as well.
yes, I can compensate the Calibur - but somehow not enough.
I think because GFP glares quite bright into the FL2 channel.
With control stains (FITC & PE) I can compensate both channels quite well - but if measuring GFP cells I get an FL2 population from the GFP cells (with the same adjustment as the control) If I compensate the "false-positive" FL2 population, PE positive cells and as well the GFP population are not detectable anymore. (they fall into the negative)
Just I got the information that GFP and PE can not be combined - but some people can do it...?
That really depends on the level of expression of GFP, as you said. For example, the CAG promoter is a big mess, you will have GFP everywhere. Also PI, if you use too much, can show up everywhere. Try ToPro3 at 1:10,000, that should help.
Thanks a lot!! I will try like you said!