Hello Everyone,
I'm conducting a plaque forming assay to detect and count Influenza virus particles. It worked well till several months early, where even my positive control doesn't show any Cytopathic Effect (CPE). I thought it was a problem related to the cells culture, so I managed to get a new vial from another team (however they don't use MDCK for the same purpose) and it still doesn't work. I changed the crystal violet, thinking that the problme might be related to that, and nothing changed. It's like the cells aren't receptive to the infection. Two weeks early i tested three different sera to grow my cells, and it's the same thing. The protocol worked perfectly before, but now i don't understand what's happening, and I really need these data.
Thank you
the first and rapid test for evaluation of influenza replication in cell culture is HA. it s better to use HA at the first. CPE is not sensitive and it s not clear for flu virus in MDCK Cells
the second point: I suggest to check all step of the cell culture and all the material should be checked such as. DMEM, FBS, PLATE, CELLS, TRYPSIN, AND ALL MATERIAL...please changed every material one by one to understand the problem. I sure one of the material or steps is not act well. and in my experience FBS is one of the most important material.
Let me know when it work it, because I´m just starting to do Flu plaque assay on MDCK cells.
Try Avicel instead of CMC or agarose. I believe you get bigger plaque size with it.
Hi Asma
Many strains of flu A should form good plaques in MDCKs and when they do, it's a lot more sensitive than HA assays (orders of magnitude). What strain of virus are you using? pdm2009 or some more recent H3N2 human strains won't plaque in MDCKs.
Just to be clear though, you're not measuring particles - you're measuring infectious units; not one and the same, as flu A will often show a particle to PFU ratio of ~ 50. The HA assay indirectly measures particles.
Avicel overlay is a good suggestion, followed by agarose. CMC overlays work, but will often give streaky "comet" plaques which can be harder to visualise.
Presumably you know to use serum-free medium with some BSA and trypsin? Titrating the trypsin is always worthwhile. The other issue that's not always obvious is that not all MDCKs are equal. You want cells that form "cobblestone" monolayers and that take a long time to release with trypsin when you pass them.
Paul
Hi Ayari,
Some avian flu strains cannot form plaques on MDCK, e.g. Mexican-like H5N2 viruses.
If you are working on avian influenza viruses, CEF is highly recommend.
David
Hello Everyone,
Thank you for your answers. In fact the problem was solved when I purchased a new TPCK, I think the TPCK we had is somehow old.
Asma
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