Currently.I am doing plaque assay on vero cell to detect the virus titer of lung homogenate from Sendai Virus infected mouse.The vero cell on the 6-well plate looks good before infection. But after inoculated with 200ul lung homogenate and incubated for 15min, the vero cells tend to shed from the plate, and the less dilution the worse(please see the picture attached).On the contrary, the mock well looks good. As the shedding is so serious and sometimes it is hard for me to calculate number of the plaques accurately.I wonder why this phenomenon happen? How to solve this problem?
Do the monolayers you loose sit without media for longer?
I've seen this with BHK cells in that the longer the cells are allowed to sit between aspirating medium and adding innoculum/plaque media the more they dry out and shed off the plate. So now I transfer media in half the wells at a time instead of pulling it off the entire plate first.
Thank you Adam! It takes me roughly 2 min to aspirate the medium and add innoculum. I guess maybe you are right and I will try your method tomorrow to see what will happen.
Thanks Andrei. But I used the agarose as the overlay every time I performed this assay.
I redid the plaque assay for two mice lung homogenates from the convalescent period of Sendai virus infection.
I tried the following methods to ensure getting a better plaque result this time.
1) I seeded the cells on the 6-well plate two days before performing the plaque assay to ensure the vero cells adhere the plates more firmly.
2)To avoid the cell from drying out, I transfered media in half the wells at a time instead of pulling it off the entire plate just like Adam did in his experiment.
3) I used disposable blue tips(1000ul) instead of using the yellow ones(200ul) to aspirate the medium so that the aperture is large enough for shearing forces not to damage the monolayers.
However,as you can see from the attached file, although it seems that it's much better than last time, yet the cell monolayers in some wells are still incomplete. I really have no explanations for this!
Hello, Have you solved your problem? Because We observed the same problem for Influenza virus titration. We wondering if the T° of the agarose layer could be the problem?
Hi Nathalie, I don't think the temperature of the agarose would be the problem.Because I noticed the cells detach from the plate before placing the agarose layer. I guess I've solved the problem, not that perfect though as some of the cell layers are still not intact. I did several serial dilutions for the each sample and get an average titration. For example, assuming the real titer for sample A is 2*10^3 PFU/ml, I did 1:10 ,1:100 .1:1000 dilution, then I probably get 186, 17, 1 plaques in those different dilutions respectively. So the range of the titration would be somewhere between 1*10^3-1.9*10^3. So in this case I believe 186 and 17 would make more sense so I will present the final titration as 1.8*10^3 PFU/ml (the average of 1.86*10^3 and 1.7*10^3 and just ignore the third dilution).Anyway, I think the deviation is acceptable.
Would you mind telling if you use trypsin in your plaque assay protocol for Sendai? If you do, do you add it into the agar overlay? what is the final concentration?
Thanks
Hi Dacquin, I do add trypsin in the agar overlay.The final concentration is 2ug/ml.
Hi Wenfang Xie,
I have similar problem with my HEp-2 cells which I used for RSV titration. Most of the time, the cells shed around the edge of the 24 well plate (top or bottom corner). I found that placing my infected plate in a small CO2 incubator sometime is quite helpful as I guess this might due to CO2 distribution inside the incubator especially if it heavily used (open frequently).
I think because you only use 200ul, it is not enough to cover the plate and most likely set in the corner of the plate so kill all cells there. Try to shake and distribute the virus every 5 minutes. Or you can use the 12 well plates for better coverage of the well surface
The toxicity induced by lung homogenate may cause monolayer disruption. You may clarify the homogenate at a higher speed followed by filtering the homogenate with 0.2um centrifugal filters prior to dilution for plaque assay.
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