3 replies to this topic

[leochicaybam]

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

[Functional Screens]

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

[Piersgb]

Why not use the 0.22um filter?

[laurequillo]

Piersgb, on 13 December 2010 - 04:40 AM, said:

Why not use the 0.22um filter?

you will lose a lot of virus