I would be very grateful if anybody could suggest any ways to improve the detection of GFP transduced cells injected intravenously. Specifically the cells are myeloid progenitors and are transduced lentivirally. Perhaps somebody could suggest a good antibody? Perfusion technique to resolve background autofluoresence? Improvements top lentiviral transduction methods? or perhaps time points for migration of cells to specific organs and IV injection?

To detect GFP in vivo, you will perfuse the target organ with a solution of paraformaldehyd 4% in saline.

Thanks Praud C. I have of course been perfusing with 4% parafomaldehyde. In fact perhaps you could help me further, what volume of PFA do you perfuse with into a 6-8wk old C57BL6 mouse?

You will perfuse during 3 minutes.

There's GFP and there's eGFP.

Enhanced GFP is brighter and thus easier to visualise. We now have a PET scanner which allows us to visualise FITC and GFP (and others) in-vivo whilst the animal is alive. How cool is that?

Frasermoss-

I had a quick look on PubMed ("PET, GFP, mice") and it only spat one paper out (see below). If you replace "mice" with "imaging" you get a few more but not all of them have used the PET to detect GFP. All refs are from 2005, so it's pretty new stuff.

Hung et al. Clin Cancer Res. 2005 Nov 1;11(21):7749-56.

I'm thinking about using this to track the expression of a virus delivered bicistronic cassette that expresses both shRNAs and eGFP. I'll post any other publications that may be more relevant once I've spoken to the group that is setting up the PET scanner.

Sounds great Parvoman, so is this a special Pet scan for mice or can a human version be used also do you know?

The version we have is used for small animals but it's the same technology as that being used in hospitals. Resolution is good in tissues that contain a lot of liquid (blood, water etc), but poor in say the lung where you have large air spaces.

New protocol listed for this technique - GFP cells in a mouse. complete with detailed instructions.

http://www.scientistsolutions.com/science-protocols_displayprotocol_bb5a2e38-ead7-4530-b1f5-b472133d2045_db334a4c-6a86-48b1-a154-10e35d9800a7.aspx

Rb