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GFP and PE


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#1 Iwenim

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Posted 01 March 2011 - 07:40 AM

Hello all,

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 !!!
Iwenim

#2 Rsm

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Posted 01 March 2011 - 11:39 PM

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.
I got soul, but I'm not a soldier

#3 Iwenim

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Posted 02 March 2011 - 01:46 AM

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...?

...
thanks!

#4 Rsm

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Posted 03 March 2011 - 12:06 AM

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.
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#5 Iwenim

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Posted 03 March 2011 - 03:56 AM

Thanks a lot!! I will try like you said!
:)




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