Contamination by packaging cells? - (Nov/12/2007 )
I don't know if anyone have come across with this idea, but I read that is a 10% chance. If so how can I confirm it? I used a retroviral system to infect stromal cells using an standard protocol in which there's an step that the virus supernatant is filtered before putting on my target cells. These cells were used in a xenograft and I got BIG tumors!, even in my controls(? 
).
Is there a way to "clean" my cells or at least confirm the presence/absence of Phoenix cells?
Any help or suggestions will be really appreciated.
Thanks
I am not sure what protocol you have used. But I guess there is another step of purification needed before you could use the virus for invivo stuff. I am guessing this from my experience with lentivirus.
Check this protocol
[attachment=3822:LV_produ...ion_2006.pdf]
).Is there a way to "clean" my cells or at least confirm the presence/absence of Phoenix cells?
Any help or suggestions will be really appreciated.
Thanks
I contaminated some BM cells with phoenix cells once (I had forgotten to filter my SN)...You can run a quick FACS and look at FSC vs SSC to see if you have a contamination (assuming your cells aren't identical to phoenix in size of course
) Clare
Scolix, thanks for your lentiviral protocol. We're using a shorter version without the sucrose step.
Clare, that's a good idea,I can certainly sort the cells. I'm working with primary epithelial and stromal cells. I think for the stromal cells wouldn't be a problem. Anyway thanks for your suggestion.
Someone in the lab told me that I can treat the cells with hygromicin to see if the "Phoenix cells" survive, or do western blot for SV-40 (?). Any comments on this? or other ideas?
Thanks
Clare, that's a good idea,I can certainly sort the cells. I'm working with primary epithelial and stromal cells. I think for the stromal cells wouldn't be a problem. Anyway thanks for your suggestion.
Someone in the lab told me that I can treat the cells with hygromicin to see if the "Phoenix cells" survive, or do western blot for SV-40 (?). Any comments on this? or other ideas?
Thanks
Hi, Phoenix cells should express murine CD8, so you can check for its presence.
... one more note- what is the origin of your target cells (mouse/ human) ? If murine, then you can design some PCR test which is able to distinguish some human gene from murine since Phoenix as you know are human origin.