Hi,
Traditionally, you need to look at infectious virus particles, rather
than expression of GFP. However, I am not sure if you harvested virus
by lysing cells from your 'experiment', and then used this as an
inoculum for titration. If so, you are technically right. The dilutions
are best chosen to represent 50-500 cells infected in a 96 well plate
when this is all done visually, for example by immunostaining infected
cells. By FACS sorting, you will need to cover a range that is within
the sensitivity of the detection settings. You may also possibly count
fluorescent foci by titrating triplicate wells for each virus dilution
in a 96-well plate. The Reed & Muench method is commonly used for virus
titration, and too few infected cells or too many of them will skew the
graph (depending a lot on your detection method). However, with each
ten-fold or five-fold dilution of virus you should see a corresponding
increase in number of infected cells. One extremely critical issue is
uniform cell plating for infection and this can considerably affect
infection efficiency. Try avoiding wells along the edge of the plate as
these yield unreliable values, and rock those plates every 15
minutes!!!
Hope this helps you..
Those magnificient viruses bring us all together....
A
Dexter Chun wrote:
> Hi all,
>> I'm trying to figure out the basis behind the 'right' calculation for viral
> titre (u/ml) based on serial dilution of viral supernatant on NIH 3T3 cells.
> My transducing efficiency is assessed based on eGFP expression by
> FACS. Hence the graph is constructed using %eGFP v serial dilution fold.
> I've been told to choose the point on the best linear part of the curve, *
> intuitively* the midpoint of the straight portion to get my titre (u/ml).
> Is this the right or best method?
>> What is the right way to go about the graphing? By using all the data
> points, I have a 'weird' curve. Is the viral titration meant to result in a
> linear correlation theoretically?
> a) do i remove the extreme point, and plot a linear curve based on remaining
> ones
> b) or is there some mathematical conversion of points OR the axes
>> Thanks.
>> Dexter
