I need to concentrat my lentivirus (2nd generation) to transduce the T cells. I can have reasonable amount of total virions estimated from the crude titration, but the concentrated virus titer is way lower than the expect. The concentration efficiency is estimated as 4-5%. Is there any suggestions for me on how to improve my concentration efficiency?
My protocol for concentration says:
1. centrifuge @ 1500G 4 degree for 10min to remove the cell debris
2. layer the supernatant over the 10% sucrose ;
3. centrifuge @ 10000G 4degree for 4 hrs
4. remove the supernatant and dissolve the virus pellet with RPMI medium
5. store @ -80 degree
I too came across a protocol in a prestigious journal that says this works. I can't say it does in my hands. If you don't have access to an ultracentrifuge, use PEG precipitation. That will work well at 10,000G. With free virus I had no such luck.
Hi, Juliette (Delhove) made some awesome lenti preps with titres higher than 1e10, paper here (open access) https://www.nature.com/articles/srep11842
Raj (Karda) has also made some very high titre/concentration preps, paper here (open access, again) https://www.nature.com/articles/s41598-017-06696-w
Hope these help, otherwise drop me an email & I'll try to put you in touch with someone who might be able to advise. BW, Simon
Thank you John Schloendorn for your answer. I thought we might read the same paper^_^. Even I myself am eager to try the method you mentioned, but I was told that my protocol can not be modified. I will struggle with the present protocol to see if I would have luck with it.
Anyway, thank you to relieve me with your experience.
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