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Determining viral titer

viral titer

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#1 sssss

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Posted 15 July 2013 - 04:39 AM

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.

#2 bob1

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Posted 15 July 2013 - 01:34 PM

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)).

#3 sssss

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Posted 15 July 2013 - 09:25 PM

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

#4 bob1

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Posted 16 July 2013 - 12:40 PM

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.


#5 sssss

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Posted 16 July 2013 - 08:47 PM

Thanks a lot for your help....




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