Hi
Could someone please tell me how to consistently get PR8 to replicate in MDCK cells in culture? What are the fetal calf serum requirements? How about inlcusion of trypsin in the media? Is the virus very sensitive to pH changes of the media? I was getting good replication in DMEM with 5% FCS w/o any trypsin,but suddenly viral replciation has gone down. The starting viral stock itself is good. So please let me know if there is a reliable protocol. Please keep in mind that I am also looking at the ability of different antibodies to inhibit replication. So I do not want components that may destroy the antibodies. Thanks in advance for your help!

I'm interested in this too. A coworker is trying to get some clinical strains to grow in culture to infect eggs and I'm using the egg harvest to do a microneutralization. The RT PCR and HA results are good from the harvest fluid but the MN isn't working well. Of course PCR results don't mean infectious virus, just that the RNA is there, I'm not sure if the HA requires infectious intact virus to work or not.
We started using some MDCK cells we had in house and found out another company location was using some MDCKs from Sigma for influenza that are apparently different. I did get better results with those on a side by side comparison of both cells but it's still not that great.
I am using DMEM with 1% (I think) BSA and TPCK trypsin (can't remember conc. 2ug/ml maybe?) for the MN. I was using DMEM for growing up cells but coworker was using MEM so I switched to see if it will help. I'll find out tomorrow, but with the MN there are many more things that could be involved (fixation, primary and secondary antibodies, etc).
I'm not sure if what we're doing will help with PR8, if you've got any ideas for me I'd be happy to hear them.