I am trying to establish a plaque assay for the H1N1 strain but can not make it after multiple tries. MDCK cells were used.
Media I used for H1N1 growth and plaque assay stated below (for 50mL)-
DMEM(2x)-46mL
7.5% BSA-4mL
Antibiotics-500uL
Trypsin (0.25%0- 50uL
I am keeping all plates for 72h. Looking for some opinions, please.
Preprint Influenza virus plaque assay v1
Please see this protocol.
A few things from what you've written -
You need to consider the exact strain, or at least lineage of "H1N1". Many of the older human isolates of the 1918 lineage H1N1 plaque fine on MDCKs at 37°C. The lab workhorse PR8 for instance. Ditto for the classical swine lineage H1N1s. The same cannot be said for many of the pdm09 lineage H1N1s though, where MDCK-SIATs are needed to match the receptor preference of these viruses. Doing the assays at 35°C for longer also helps (though for us, longer = the 72h you're using).
You also don't say what overlay you're using. We mostly prefer Avicel, though agarose can work well. Carboxy-methyl cellulose will also work, but you can get streaky "comet" shaped plaques which are harder to count.
Final thing that occurs - if the assay is trying to work, titrate the trypsin and/or try an alternative source.
Good luck!
Paul
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