Any advantage on sonicating fibroblast cultures to release virions, as opposed to repeat freeze/thaw cycles? Whoever may have routinely sonicated CMV-infected fibroblast cultures, what sonication settings to use (probe type, volume of culture, pulse cycles, intensity)?
Hi Luiz,
Each method has its own pluses and minuses.
With the sonication, the problem is to use sufficient energy to disrupt the cells, but not to a point where you will destroy the virus. Freeze-thawing may have the same effect (although I tested once the loss of titer upon 4xfreeze-thawing and it was remerkably low - but this was on a purified virus stock in Virus standard buffer, and virus from cell lysates may act differently). In my lab, we centrifuge the cell pellet for about 10' in a GSA rotor at 6000rpm and dounce it in prechilled douncers in a volume of about 7ml (just like Katrin suggested).
This way we do not hurt the virus much, but pop the cells open and are able to get the viruses out of the cells.
If you use lab strains of CMV (e.g. Twone, AD169), they are not very cell associated and you will obtain ample amounts of virus from the SN (and here you do not need sonication, douncing or freeze thawing, since the virus is released already). Centrifuging the SN for 3h in a GSA rotor at max speed will pellet the CMV and allow you to concentrate this part of the virus. Clinical strains (e.g. TB40E, TR, Merlin etc...) are much more cell associated. Here you will get almost all the virus in the pellet and the rule is - the more clinical the strain, the more cell-associated it is (i.e. Merlin is more cell associated than TB40E, TB40E is more cell-associated than AD169).
Normally we pool the virus reased from douncing with the virus from the SN, to maximize the yield from our cells. In case of interest, I can send you our protocol for virus purification.
Freezing/thawing simply kills the virus. You lose at least one log titre at each cycle.
Sonicating is not just a matter of apparatus, intensity and time. It also depends on the nature of the cell debris you have in your sup. Your have to find your best working conditions by yourself I suppose. Keep in mind that sonication should be mild and in ice. You are aware of course that you should avoid formation of aerosols (risk to infect yourself), so try to wrap around your tube and the sonicator tip some aluminum foil.
Hi Luiz,
Each method has its own pluses and minuses.
With the sonication, the problem is to use sufficient energy to disrupt the cells, but not to a point where you will destroy the virus. Freeze-thawing may have the same effect (although I tested once the loss of titer upon 4xfreeze-thawing and it was remerkably low - but this was on a purified virus stock in Virus standard buffer, and virus from cell lysates may act differently). In my lab, we centrifuge the cell pellet for about 10' in a GSA rotor at 6000rpm and dounce it in prechilled douncers in a volume of about 7ml (just like Katrin suggested).
This way we do not hurt the virus much, but pop the cells open and are able to get the viruses out of the cells.
If you use lab strains of CMV (e.g. Twone, AD169), they are not very cell associated and you will obtain ample amounts of virus from the SN (and here you do not need sonication, douncing or freeze thawing, since the virus is released already). Centrifuging the SN for 3h in a GSA rotor at max speed will pellet the CMV and allow you to concentrate this part of the virus. Clinical strains (e.g. TB40E, TR, Merlin etc...) are much more cell associated. Here you will get almost all the virus in the pellet and the rule is - the more clinical the strain, the more cell-associated it is (i.e. Merlin is more cell associated than TB40E, TB40E is more cell-associated than AD169).
Normally we pool the virus reased from douncing with the virus from the SN, to maximize the yield from our cells. In case of interest, I can send you our protocol for virus purification.
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