[Virology] Re: viral titre determination (calculation) by limiting dilution

Dexter Chun dexter81 at gmail.com
Wed Aug 16 23:46:18 EST 2006


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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