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Lentivirus toxicity


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#1 leochicaybam

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Posted 30 October 2010 - 01:57 PM

Hi everyone,

Im 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 didnt discard the pellet because I was afraid to lose the viruses...


Any suggestions???

Leo Chicaybam

#2 Functional Screens

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Posted 28 November 2010 - 01:16 PM

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

#3 Piersgb

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Posted 13 December 2010 - 04:40 AM

Why not use the 0.22um filter?

#4 laurequillo

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Posted 16 December 2010 - 04:59 AM

Why not use the 0.22um filter?

you will lose a lot of virus
"He must be very ignorant for he answers every question he is asked" Voltaire

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