I am doing a plaque assay for influenza virus. am dealing with MDCK cells. After they formed a complete monolayer, they were infected with WSN virus for 1 hr at 37 C. Thereafter an agarose overlay were added to the monolayer and then the plates were kept at 37 for 3 days. The cells then fixed for overnight with formaline, and then stained with toluidine blue. After staining there are some empty unstained spaces at some places, it seems that my cells are detaching after staining as it shown in the uploaded image. I tested the possible reasons like the effect of agarose layer by culturing two plates with and without agarose layer and also the drying effect by increase the amount of the virus culture from 450ul to 2ml just to cover the well the problem still exist If somebody knows what to do next, please suggest how I can improve this method.
thanks in advance
Hi Zaid
Try to decrease the agarose concentration or you may want to try carboxymethyl cellulose (CMC) 2% in Medium insted of agarose. After virus absortion add CMC 2% [we add 500 ul CMC 2% to a well (12 well plate)]. Remove by Vaccum or pippet. fix with paraformaldehyde for 15 min and then stain. I think the main problema is the agarose.
After going through the picture, I think it is a case of drying of the monolayer.
Decrease the time interval between discard of the culture (virus culture used for adsorption) and addition of agarose overlay. Again make sure that the agarose is cooled upto 50 degree C before addition to the plates.
I do not think agarose has any role in this.
Also another thing you can do is inoculting the virus culture at 80% confluency and then allowing it for 3 days. This helps in preventing overgrowth of cells. However, as per the pictiure attached by you I do not think confluency is the cause of problem of detachment.
Thanks Martin, I tested the effect of the agarose it doesn't has any negative effect on the monolayer because I used 2% agarose and then mixed it with equal volume with MEM medium so its quite porous. appreciate your suggestion.
Thanks Laxmi, I think you suggestion make sense to me because the degree of health of the monolayer have a direct effect on the positivity of the result plus the the time between the time interval between the culture discard and the adding of the agarose.
appreciate your suggestion.
am going to try washing my cells after the discard of the medium with PBS supported with Ca and Mg this is one way to support cells attachment which might helps because I used to wash with PBS only
Thanks Martin, I tested the effect of the agarose it doesn't has any negative effect on the monolayer because I used 2% agarose and then mixed it with equal volume with MEM medium so its going to be 1% so its quite porous. appreciate your suggestion.
Thanks Laxmi, I think your suggestion make sense to me because the degree of health of the monolayer have a direct effect on the positivity of the result plus the the time between the time interval between the culture discard and the adding of the agarose.
appreciate your suggestion.
am going to try washing my cells after the discard of the medium with PBS supported with Ca and Mg this is one way to support cells attachment which might helps because I used to wash with PBS only
Looks like either your agarose is too warm when adding or could be that your volume of fixative/stain is too low and not adequate staining. would suggest trying a different overlay (CMC or Avicel) since is liquid at all temps and will not peel off monolayer.
thanks Thomas, I could overcome this problem it was actually with degree of cells monolayer healthiness
Hi Zaid, I was wondering if you found out what was the problem causing the cells to detach?
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