Do you really understand the way “titer” “work” in case of transduction of Hela? - (May/02/2010 )
I had never figure out the way “titer” “work” in case of transduction of Hela.
It seems titer is a some kind of concentration of virus,which is actually important to the transduction efficiency of like Hela cell,instead of the totol number of viruses.
Which means even if you infect hela with a proper totol number of lentivirus in a large volume of supernatant with relative low concentration(titer),you still can’t get a satisfying transduction efficiency.
If that is true,it’s weird,isn’t it?
How can you image that in case of a low titer,there is a lot of virus out there in the supernatant but just can’t enter the cell,while they can when the titer(concentration) is high.
So could anyone tell me why?Or where is my misunderstanding?
Titre is the strength of a solution of anything as determined by assay. In the case of viruses the titre is important as infection often will not result from one virus particle, usually several are needed per cell to produce an active infection.
Think about your infections this way - if you take 20 balls and put them into an office so that they are all bouncing around, and then you enter the office, your chances of getting hit are quite good. Now take the same 20 balls and bounce them around in a basketball court. Your chance of getting hit lowers drastically. The titre is effectively the same; you are keeping the total amount of balls (virus particles) the same, just in a smaller volume (higher titre), so the chance of a hit (infection) increases.
bob1 on May 3 2010, 08:30 AM said:
Think about your infections this way - if you take 20 balls and put them into an office so that they are all bouncing around, and then you enter the office, your chances of getting hit are quite good. Now take the same 20 balls and bounce them around in a basketball court. Your chance of getting hit lowers drastically. The titre is effectively the same; you are keeping the total amount of balls (virus particles) the same, just in a smaller volume (higher titre), so the chance of a hit (infection) increases.
Thank you!you are right.
to What degree do you think the titer will fall if I double the volume of medium which I used to collect the viruses that were secreted from packaging 293T cell and given the totol virus number doesn't change.Will it fall a half or somewhere else?
Cause I double the medium I can collect viruses for 48h while it won't turn yellow,otherwise it will,and this are thought to harm the virus.
I don't know how the titre will be affected, I suspect though that it will be non-linear, possibly some sort of logarithmic relationship?
bob1 on May 5 2010, 08:42 AM said:
yes，non-linear，I also think so
You could always concentrate your virus stock with a centricon filter, or more simply (and cheaply), just ultra-centrifuge the stock and resuspend it in a small volume.
For a couple of our HSV mutants, not much virus is produced, so I wind up having to use scads of cells to produce it... which means tons and tons of media. I just pellet the hell out of it in the ultra and suspend it in a small volume of buffer... You could potentially concentrate it as much as you want. I've gone from 1x10^7/mL to 4x10^9/mL by doing this. It makes it possible to infect with high titers for experiments.
For HSV or baculovirus, we use ~80,000g for an hour or (preferably) lower g for longer. The pellet is like a rock, but soaking overnight at 4C and some light sonication fixes that. Trasnduction can take some seriously high titers... on the order of an MOI of 25-100 sometimes. With that, we can get close to 100% transduction.