Plaque assay for murine cytomegalovirus - (Jul/16/2009 )

Hi,

Does anyone had experience in doing plaque assay to measure the titer of MCMV? I have been trying NIH3T3 in different condition (cell density, infection days), however, the confluent layer didn't form plaques.

Do you think I should switch to primary mouse embryonic fibroblast? I checked Millipore and ATCC are also selling MEF cells. Which company do you recommend and what's the difference between different MEF cells (antibody resistance, drugs treated, different passage etc)? Is feeder cells equals to MEF cells?

If the titer is just too low, how can I cencentrate the virus? Is there any suggested protocol for MCMV?

Thank you very much for your kind advice.

My lab works on MCMV and we do plaque assay to measure infectious virus all the time (we usually use primary MEF, but I have used NIH3T3 and BALB/c 3T3 successfully also). Can you give me a step by step of your protocol and I will see if I can help (PM me your email address if you prefer and we can communicate that way).

leelee on Fri Jul 17 06:09:57 2009 said:

Hi,

I am sort of new here. We have been working hard on viral plaque assay for murine cytomegalovirus using M2-10B4 cell line with no positive outcome. We have now switched over to NIH 3T3 and MEF lines to try if they work. Generally what overlay would you recommend, the agar or methyl cellulose. Right now I am growing both the cell lines in a T75 flask using DMEM supplemented with FBS. Later I am planning on to seed 10,000 cells into each well of 24 well plate and then grow for 1 day and then add virus (about 100 uL of each dilution) allow adsorption for 1 hr at 37 C and then wash with PBS and add overlay medium and after 5-6 days add the fixing solution and then remove the overlay. What do you suggest for fixing, we do have crystal violet in the lab. And if you have any microplate plaque image would you be able to share it, so that I can visualize how my plaques should look. I have been trying really hard to figure out a way for this to work, which is a crucial point for the project to thrive and move. Any help would be whole heartedly appreciated. And also let me know know if someone has tried X-Gal staining earlier.

Thanks,
Vamsi.

Hi,

I'm also new here in this forum. Currently I work with murine cytomegalovirus, but it seems that the method by using plaque assay to determine the virus titer is troubling...

It is quite difficult to distinguish the plaques because you have to use a high magnification (x200) Are you familiar with this problem??

I use NIH-3T3 cells for this titration by the way.

/Marie