Lentivirus toxicity - (Oct/30/2010 )

Hi everyone,

I´m trying to transduce mouse primary T lymphocytes with my empty vector (pLVTHM - GFP reporter). After the production of the viruses, I harvest the supernatant and centrifuge to remove cells/debris (2000rpm/10min) and filter it using the 0,22um syringe-driven unit from Millipore (PVDF membrane). However, after the ultracentrifugation step (using standard speed to concentrate VSV-pseudotyped vectors) I am obtaining a pellet (!), probably of cell debris or even proteins. Taking into account the size of the pellet, I hardly believe that it is formed only of viruses...If I ressuspend this pellet and use the virus to transduce the cells, two days later all my cells are dead. I didn´t discard the pellet because I was afraid to lose the viruses...


Any suggestions???

Leo Chicaybam

DO NOT use .22um filter, please use .45um filter before ultracentrifugation.

Why not use the 0.22um filter?

Piersgb on Mon Dec 13 12:40:52 2010 said: