Determining viral titer - (Jul/15/2013 )

Hi,
I have started with the experiments related to the transduction of Lineage negative cells with MSCV-neo vector containing my gene of interest. I am trying hard to infect the lineage negative cells but failing to do that. When I use these viruses to infect NIH3T3 cells its working fine. Please help me to determine the virus titer and also suggest some way out for my experiment.

It would seem that the titre is dependent on the cell line you used. The classic way to determine titre is to do a plaque assay, where you seed out a confluent layer of cells, infect at a range of dilutions and then count the resulting plaques. The titre is determined in a similar manner to colony forming units for bacteria.

i.e. titre (pfu/ml) = (number of plaques/dilution) x (1/volume plated(ml)).

Thanks for your reply. Can you please mail me the protocol for plaque assay.

The protocol depends on the virus, and only works if your virus forms plaques, but in general it works like this:

<*>infect cells by your standard procedure.
<*>Remove inoculum.
<*>Mix 2% LMP agarose with 2x medium to required volume (i.e. multiply number of wells by volume) to give 1% agarose and 1x medium.
<*>Layer over infected cells
<*>Incubate for desired time (again, virus specific)
<*>Overlay with another layer of agarose/medium containing 0.04% neutral red (stains living cells, making plaques easier to see).
<*>Count plaques formed - wells of a 6 well plate with 100-500 plaques is the range to be counting. Higher than that and it gets difficult to count, lower is inaccurate.

Thanks a lot for your help....