I have infected A549 cells with two different types of Adenovirus. Today is the 3rd day post-infection. I would like to isolate viral DNA and also do viral titration but only later, someday next week. I was instructed to collect the medium (which consists of viral particles and swimming cells) and freeze it in 15 mL tubes at -80 C. The rest of the cells which are still attached to the plate I should treat with trypsine, centrifuge and dilute with PBS and just put to freeze at -80 C. Is there perhaps some better way to preserve the material for later usage?
If you are planning to re-culture your frozen cells it is better to resuspend them in freezing medium (commercially avaliable but easy to make yourself, just 10% DMSO in ordinary media). But in isopropanol-ice pack ("Mr. Frosty") so that the samples doesn't cool to quickly. Freezing media is slightly toxic so make sure to put them in the freezer ASAP, also when thawing it is important to quickly get rid of the freezing medium.
I use DMSO to freeze cell culture. you can use too.. First treat with trpysine and than centrifugate (1100 rpm) to remove trpysine that can affect cells negativly. remove supernatant and add medium and %10 DMSO. If you put -80 directly, cells can be damaged... First you can put 4 C for 1 hour, than -20 C for 1 hour and lastly -80 C... The main object for these protocols are preventing fast crystalization of water that will damage cells. Fast thawing also significant for cells, also you have to change medium because of toxic effect of DMSO.
I suppose that you are trying to produce more adenoviral vectors. I have experience with HEK293 cells and when I want to collect my adenoviruses for future experiments, I take the supernatant and detached cells in one tube, and left in my flask/plate about 20% of the total volume of medium to scrape the cells still attached without trypsin, only scraping. Then I put it in another tube for maintain at -80 °C. before I use the cells scraped, If what I want is to use the adenoviruses than contain inside for future infections, I do 3 freeze/thaw cycles to liberate the viruses and now be suspended in the 20% of the total volume and it will be more concentrated, because the most part of them will be inside the cells that still not have been lysated.
There is some references that recommends you add glycerol to a final concentration of 10% to your extracts, to protect the adenoviruses.
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