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Low titer Lentivirus: concentrate or add whole volume?


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#1 cancerous

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Posted 07 September 2012 - 09:18 AM

Dear all,

I produced Lentivirus for shRNA clone with which I would transduce to human cell line growing on the culture dish. However its titer seems to be low, so I am considering to concentrate it. But someone says there is some loss when I concentrate it. Then, I want to add whole volume of virus to the cells for transduction. I think volume does not matter but the number of active virus particles is important.

But I just want to make sure if:
Virus in much volume can be infected less efficiently than virus in little volume if the same number of virus particles present or the iinfectivity is the same in theory.

All the best,

Cancerous

#2 Hazelnut

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Posted 10 September 2012 - 05:30 AM

Hi Cancerous,

The total volume that your virus is in DOES make a difference to the efficiency of infection or transduction. The lower the volume, the higher the rate of infection simply because your cells will be more likely to come into contact with virions. So keep your volume as low as you can.

It's true that pelleting in an ultracentrifuge damages your virions a little, and so overall you will lose a certain percentage of the infectious virus particles. But to be honest I find that if you pellet your virus and resuspend overnight at 4C on a gentle shaker, with minimal pippetting, they will be fine and your titre significantly higher.

If you are using, say, a 6cm dish, and you have 10 mL of virus supernatant, I would say you're better off pelleting it down to 1mL and using it all rather than trying to chuck all 10mL onto your cells.

good luck!

#3 zodiac1505

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Posted 18 October 2012 - 09:24 AM

I agree with Hazelnut on this. It is better to concentrate your virus and then use it. Once you spin them down, add 100 micro liter of PBS along the side to the tube without disturbing the pellet and let it stand overnight at 4 degrees. You can then resuspend the pellet the next day and use the suspension for titrations and infections.

Since you mentioned that your titers are low, you might want to try out other transfection reagents.




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