Naked Science Forum
Life Sciences => Cells, Microbes & Viruses => Topic started by: Garglaes on 10/11/2015 01:02:53
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Currently in my lab we use a few cell lines ( Vero 76, MDCK, LLCMK2, HEP2 ) for diagnostics and research of respiratory viruses. We have noticed that out cells are not as receptive towards our respiratory virus samples as they do not show CPE for any of our samples. We performed a test with our viral controls to see if our cells would still demonstrate CPE with known viral titres as well as knowing which cell line was receptive to which virus and therefore know what type of CPE we should be observing. Our results demonstrated that our HEP2 cells infected with RSV started to detach around day 6 and when we performed immunocytochemistry, no fluorescence was noted. For our influenza samples, our MDCK cells demonstrated 3+ CPE around day 9, while previously they were harvested at day 3 with 3+ CPE, and the immunocytochemistry was low around 1-2+ CPE.
Also, for Influenza A we usually see grape-like group of cells as a positive CPE followed by total destruction of the cell monolayer, however, now we see CPE as the cells forming darker cell membranes and slowly losing the inside of the cell, while still remaining in monolayer or sometimes a monolayer with small holes of cells missing. It would seem as if the CPE remains stunted, one can tell the cells are unhappy and you see the darkening of the membranes as small granules if you go on higher magnification, yet they remain stunted and do not have a degradation of the cell monolayer as we usually observe when MDCK cells are infected with Flu A.
We are thawing the same frozen lot of cells at the same passage as we have previously when we infected cells and had no problem with CPE, the same media and the same viral supernatant is used with the same protocol. We thought the problem could be the media and its variables, we use 10% FBS ( same lot as when cells demonstrated CPE ), E - MEM ( again, same lot ), 1% Antibiotic-mycotic, 1% non-essential amino acids, 1% sodium pyruvate. We borrowed some powdered media and prepared it from scratch to see if the media was the variable affecting the cells, as well as obtained a new set of non-essencial amino acids from a different company. Any ideas as to why we have little/different CPE in our cell lines ?
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I have heard of cases where labs were using different cell lines than they thought they were using. This has cost a lot of time & money. You can't really tell without sequencing the cells.
There are lots of potential causes - they were sent the wrong cell line, the culture was taken over by another strain in the lab, etc...