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
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Lentivirus toxicity
Started by leochicaybam, Oct 30 2010 01:57 PM
3 replies to this topic
#2
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Veteran
Posted 28 November 2010 - 01:16 PM
DO NOT use .22um filter, please use .45um filter before ultracentrifugation.
#3
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Enthusiast
Posted 13 December 2010 - 04:40 AM
Why not use the 0.22um filter?
#4
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The Goddamn Batman!
Posted 16 December 2010 - 04:59 AM
Piersgb, on 13 December 2010 - 04:40 AM, said:
Why not use the 0.22um filter?
you will lose a lot of virus
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