Try using agar (usual medium) instead of CMC.   Clearly your overlay is toxic.

Dear Peter,

Thank you. I have tried with agar once. Cells were detaching from corner of wells. Then lower the agar concentration. Cell detaching and some disturbance in cells were present in lower concentration also. What could be the reason? Still too high concentration of agar? Or something else? 

Image attached

Dear Abdullah.

Sometimes I am facing exactly the same problem, e.g. for Usutu virus. It happened, when the EMEM-CMC was prepared several days before, even 1-2 weeks. I always use as much as possible fresh EMEM-CMC.

Also, this may happens when cells are a bit dried. I do not know your protocol, but we removew the inoculum before the overlaying. This is a critical step, ifyou process more samples/plates. The laminar flow can very quickly dry the monolayer. Vero cells are quite robust, but you have to be careful and keep them dry. I always cover the palte with the lid, to protect the mnolayer from the air flow. But even when I try as much as possible, sometime I have to discard a plate, or several plates because of this.

Very important is the disolving the CMC. I disolve the 2% CMC for 1-2 days in refrigator. You have observe the medium carefully, because if the CMC is not completly dissolved, it may appear as a "jellyfish" in the viscous EMEM :).

You asked this question 5 month before. Did you solve the problem? I am asking, because I am starting with the standardization of plaque assay for RVA. How is it work on MA104 cells, the plaques are clear?

Thank you very much.

Tomas.

Dear Thomas Csank,

Which cell line do you use for performing Plaque assay and/or TCID50 for Usutu virus in your lab? Please, I would also like to know the dpi to get a good titer.

Thank you very much.

Eric.

Dear Abdullah

Have you try to maded it with another cell line? I used BHK-21 for plaque essay with flavivirus and i don´t have any problem.

I thing you have to reduced the concentration of CMC to 0.8%. It permit the gas exchange and reduce the cellular stress.

Dear Tomas Csank , thank you. We didn't have MA104 cell so rotavirus plaque was not visible in vero cells. I didn't find any reference who worked with vero cell for rotavirus plaque. However cells were okay later with agarose overlay.

Recently I am working with influenza plaques on MDCK cell lines. Here also CMC is showing little effect on cells while agarose none.