Hello , Thank-you very much for sharing the Immunoplaque assay...

Hi Ramuth,

I will suggest you to keep the MDCK cells at 90% when you start your inoculation. Fifty percent is too low for plaque assay. Good Luck!

Cheers,
Vic

Hello Vic ,
Thank-you for the valuable advice.
In fact i did not fix the cell monolayer , I will do so next time .
Confluency also i used 80 % . Will use 50 % in future plaque assay .
Thanks again .

Kind Regards ,
Vanessa

Dear Ramuth,

I am sorry that your plaque assay did not work, but you are definitely not the first one. Plaque assay is quite tricky to deal with. I put some pointers below and hope it will help.

1. There are multiple type of MDCK cells, MDCK.1, MDCK.2 and the generic MDCK which is a mix of the two. For more information, please refer to the paper below.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209442/

2. Do not let your MDCK go to 100%, since plaque assay tend to take at least 2 -3 days to finish after the inoculation of the viruses. Your MDCK might overgrown and died if your initial confluence is too high. Judging from the picture you provided, your MDCK cells died or detached before your staining.

3. Did you fix the cell mono-layer with 4% PFA? A lot of people tend to fix the cells after the removal of the agar, I personally found fixing it before removing the agar help to better retain the mono-layer structure.

4. My protocol have another twist from the generic protocol, I basically did immunohistology instead of crystal violet to visualize the plaques. It woks exceptionally well for tiny plaque.

Please let me know if you have further questions, I will be glad to help.

Cheers,
Vic