Aubrey N. Michi, thank you! We will consider your suggestions. Probably we have to increase MOI then and it will solve the problem.

Just one curiosity... For what I am aware of, I can't use A549 to plaque assay the virus I am using since it is trypsin-dependent and A549 cell line is sensitive to it. Do you know other alternative?

Thank you again!

Dear Mariana Marques,

I am happy to hear that you intended with influenza A virus (PR8). First of all, I agree with Miss Aubrey N. Michi answers.

I suggest you do optimize the cultivation condition of influenza A virus on MDCK and/or A549 cells with a suitable condition utilizing comparing the different concentration of TPCK-Trypsin (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 5.0 micrograms/ml) in different time point (0, 4, 8, 12, 18, 24, 36, and 48 HPI) and check virus titre via qPCR or western blot by testing influenza N, M, and PB2 protein. As well as optimize serum-free media before 1, 6, and 12 hr of virus infection.

Good luck.

Hi Mariana Marques Nattan Stalin Aubrey N. Michi !

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

First of all, Nattan Stalin and Aubrey N. Michi thank you for your help at the time. I eventually found out that my problem was with the media formulation I was using. Thank you anyways :)

Kezia Lim I'm not sure I understood your question. Are you doing the plaque assay in A549? Or are you using A549 to perform the experiments and then you do the plaque assay?

I usually use MDCK as the standard cell line for the plaque assay, and I use 1 ug/ml TPCK in DMEM supplemented with 0.14% BSA, and 50% avicel (cellulose). I don't have any experience with agarose, I'm sorry.

Good luck!

Hi Mariana Marques

Yes, I'm using A549 to do plaque assay. I'm well aware that usually A549 is used to take the viral supernatant and reinfect it to MDCK to observe the plaque formation, and not being used for plaque assay solely. However, previously I tried to use A549 for plaque assay and the result was better than MDCK. Hence, after several discussions, we decide to use A549 instead MDCK to treat PR8/H1N1 virus.

Nevertheless, I have to change my A549 cell as it was quite old and we faced contamination. So, we take a new cell of A549, but so far no result was produced by using the new A549. I was wondering if it happened due to the overlay media formulation. As previously I used 2ug/ml of trypsin-TPCK, 6% agarose gel, and L15 media. I looked out the other study some of them using 2ug/ml when treating A549 using virus, and I also found that the recommended concentration for trypsin TPCK for A549 is between 0.1 to 0.5 ug/ml (not really sure what type of experiment they did).

As in the question you mention that you infect A549 with A549 that you produce in MDCK. I just wondering if you have any suggestions for my formulation, especially for TPCK? Is it 2ug/ml too high for A549?

I'm very new to this type of assay. I'm really appreciate your help!

Hi Kezia Lim

Good to know that it is also possible to use A549 to perform plaque assay with PR8.

I don't have much experience with using TPCK in A549 as I normally perform experiments with a single infectious cycle. However, I would try 1 ug/ml (or less) in A549 or try a different % of agarose in the media.

* I just noticed that I told you the wrong avicel %. I use a final concentration of 1.2% avicel.