I used MDCK cells for my plaque assay for influenza virus and seeded 950,000cells/well in 6-well two days before infection. The virus was diluted in FBS-free MEM with 0.5ug/ml TPCK-trypsin. Here is my recipefor plaque:
10ml 2.5% Seaplaque agarose + 9ml 2x MEM + 40ul TPCK-trypsin(0.5mg/ml) + 1ml BSA(0.06g/ml)
(final concentration: 1.25% agarose, 1ug/ml TPCK-trypsin, 0.3% BSA, ~1x MEM)
I harvested at the third day after infection. Some plaques are clear and visible, but some are not as the picture showed below. Can anyone tell me what's wrong with my assay or virus?
Thank you!
Hi, Could you please tell me
1. Incubation time for virus adsorption in cells?
2. Before adding agarose+ media solution, have you removed the remaining virus solutions from plate?
Thanks
Hi, thank you for your answer.
1. I incubate for 1 hour at infection step.
2. After infeciton, I removed the virus medium and wash with PBS once. Then I removed all the liquid in plate before adding agarose mixture.
Which virus strain did you use? Did you sequence the genome? What was initial moi and virus dilution?
Hi, I made the virus by reverse genetics system and replaced the HA and NA sequence to california/07/2009. I already confirmed the genome of the virus used in plaque assay. The genome is right. In this plaque assay, I didn't know the initial titer of the virus. I did the 10-fold serial dilution. Every well with virus have this situation.
The plaques look countable to me... Things we do (not all at once, depends on the virus) when faced with a difficult strain of flu to plaque - drop the temperature from 37°C to 34°C, increase the incubation time from 2 to 3 days, titrate the trypsin concentration, use MDCKs engineered to have different types of sialic acid, use an Avicell overlay, immunostain the plaques. Some of our pdm09 strains are a pain and need a combination of these changes to the basic MDCK 2 day with 1 µg/ml of any old trypsin at 37°C protocol to get things half as good as your photo.
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