Hi,
Thanks for the reply. The GFP expression is an indication of cells being
infected. And the harvested virus is collected as viral supernatant from
packaging cell lines i.e BOSC23.
I think what you meant is 5-fold serial dilutions will see a
corresponding *decreasing
*number of infected cells (NIH3T3).
Is this relationship linear? Or sigmodal?
I'm ain't sure what kind of relationship to expect and hence, am having
problems picking the *'right'* titre point to calculate?
Cheers,
Dexter
On 16 Aug 2006 11:57:31 -0700, adrish <adrishsen at gmail.com> wrote:
>> 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
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