I have used my IAV plaque assay protocol (2XMEM and 2X low-melt agarose overlay to 1X dilutions with 1ug/mL of TPCK-Trypsin) for a few years, with no problems. Recently, I changed labs, thus changing from my original MDCK cell line to a new one purchased from ATCC, and I have been trying to get my plaque assays to work for several months now to no avail. The cells seem to have low adherence in the 12 well plates, they wash off when I stain with crystal violet (I have tried multiple solutions for staining, 20%ETOH in diH2O, 10% formalin, etc). I believe I can see plaques before staining, but I am not sure.
Any suggestions would be great!
Thank you!
Hii I’m also having the same issue as you currently…..am wondering did you manage to solve the problem as of now?
Much apologies that I did not provide an answer……I’m just a beginner when it comes to technical skills in lab experiments and also been having issues with plaque assay for many weeks
Thank you so much for posting your question here
Shi Yuin Chong, do you know anyone that has successfully completed a plaque assay with the cell line you are wanting to use?
I contacted my old lab/mentor and got cells from him and this fixed my problem. My new stock of MDCK cells were having an issue adhering to the plate. My old stock is doing just fine now! I would start with the cells if you have went through and checked everything else I have listed above as areas to troubleshoot.
The other thing is underseeding the cells. If you are using MDCK cells in 12 well plates I like to plate 250,000-350,000 cells per well. Underseeding is can sometimes look like this, but if the cells look ~90% confluent before you infect then the confluency isn't the problem.
I changed back to my old cell line that I knew worked, so that is my suggestion to you!
Shi Yuin Chong, also, what concentration of TPCK-Trypsin are you using? It may be too high.
Hi Jessica M. Reel
Just wondering if any of you do plaque assay using A549. Currently, I'm doing plaque assay on A549 cells but I'm facing a problematic issue where my cell always detaches. I used 4% PFA for fixing and 6% crystal violet. I don't think there's any problem with the concentration of PFA and agarose gel. So far, the only problem that I can think is might be the concentration of TPCK that is too high for A549 cells. I used 2ug/ml of TPCK, L15 media, and 6% agarose for my overlay media after infection.
Do you have any suggestions for the concentration of TPCK that is suitable for A549 cells? Also, do you think 6% agarose gel is too concentrated for this cell linage?
thank you!
Hello Kezia Lim
For the Madin Darby Canine Kidney (MDCK) cell line that I use to plaque IAV PR8 I use 1ug/mL of TPCK Trypsin and no more than 2% agarose.
I would try to decrease the amount of trypsin and also decrease your percentage of agarose.
Have you had a successful plaque assay? I realize I am getting back to you late!
V. B. Oti purchase some TPCK trypsin and add it to your overlay mixture at 1-2ug/mL of overlay. That should help with plaquing!
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