Hi Yulin, I'm not sure whether this is helpful or not, but I believe I was having the same problem before during the learning phase of doing the H5N1 plaque assay. Mine was because during performing the assay;

1. I let the cell in the well exposed to the circulating air (without covering with plate cover) for too long. For example, after I took out the PBS from the well (leaving the cell only, without any liquid covering it), I didn't cover the well with its plate cover - letting it exposed to the air while I was taking some media to be put in the same well. Because I was a bit slow before, the cells became dry and died even before I manage to fill in the well with media. So now every time I take out any liquid from the well, I will make sure I only open half of the well surface, and quickly cover the well right after I'm done. I also quickly put the media into the well to make sure the cell isn't being dry for too long.

2. Previously, I also used agarose. Since agarose easily becomes solid, I put it in the water bath while waiting for it to be poured into the well as the last step. The thing was, I didn't realize that the agarose temperature was too high to be poured directly onto the cell. Since then, I changed and started to use the CMC until now (H5N1, H1N1, DENV and CHIKV).

Since I learnt those things in hard ways (I was stuck with the plaque assay for a few months) not realizing my silly mistakes, thus I hope you would be able to overcome your difficulties as soon as possible! Hopefully, my suggestion could help you (if it is the case) :)

All the best, and stay safe! :)

Sha

Hey guys,

I am having the same issue, and I was doing pretty well until I switched to different MDCK cells, my other coworker doesn't have this issue, but I do. I had her watch over me and told me my agar wasn't too hot, and sometimes it is almost solid when I put it in, and it still happens. I'm not sure what to try next. Did you end up changing anything, and how did you fix your problem?

Hey all,

I am also having the same issues. I am working with fresh MDCK cell stocks from ATCC and have used my protocol many times, but I recently changed labs, thus changing my cell lines. But I am seeing a similar outcome as above. My cells are just washing off the plate when I dye them with crystal violet. I am open to suggestions here.

Yulin, your's look like it may be a drying out problem, I would try shaking more often during the infection period.

Hi Jessica,

I'm not sure whether it will work or not, but perhaps you wanna check on how you fix your cells? I used 10% formalin, and I realized the way I put the formalin does play some roles during the washing step. Previously I just put the 10% formalin in the center of the well, but I realized that I put drop by drop of it around the well (especially near the well's wall) - just to make sure the 10% formalin spread equally throughout the well, will produce better staining. The cells (near the wall) wouldn't detach during the washing step. Plus now I prefer to incubate for a longer period during the fixing step (more than 1 hour) or perhaps overnight at 4c. Hope this helps!

S.

Hi Dominika,

I think it is more on your technical skill because you said your coworker doesn't have the same issue as yours, right..? Hrmm... in my experience, we also have to be careful of the blowing air during the plaque assay. Perhaps you might want to not let your cells be exposed for too long during each step. Usually, I use the plate cover to cover any uninvolved well while working on the other well, and quickly put media/PBS/virus inoculum after discarding what was in the well before - do not let the cells dry for too long. Hope this could help you to solve your problem! :)

S.