Multiple nuclei?? - cell morphology - FaDu (Nov/28/2010 )

Has anyone seen this before? It's FaDu (pharyngeal tumor cells). If you look at the second picture, there seem to be multiple nuclei inside a cell... Those cells eventually stop differentiating and die/detach. Any input/suggestions would be appreciated!

Senescent cells.

bob1 on Sun Nov 28 22:59:24 2010 said:

Why do they senesce so prematually? I think they are definitely under stress, but can't figure out what it is. Have you had a similar problem? If so, can you please tell me how you solved it? Thanks!

Sorry, haven't used the cells at all, but those are definitely senescent.

bob1 on Mon Nov 29 22:40:13 2010 said:

So there is no way to keep passaging them then??

No, senescence is permanant. What are your culture conditions? What sort of sub-culture ratios are you using?

bob1 on Tue Nov 30 22:27:30 2010 said:

I used DMEM + 10% FBS and 1:3 subculture ratio. I tried it with or without pen/strp. By the way, I did mycoplasma test yesterday (PCR method), and got very strong bands... But I am still not sure if it was mycoplasma or my technique that caused premature senescence. I am doubting my technique because I used to culture MDA-231 which grew fine, but I found many of them attached but didn't quite flatten. I don't trypsinize my cells for longer than 2-3 min, and I always neutralize trypsin with an equal volume of medium before seeding into a new flask or plate. I also tried spinning them down to remove any trace of trypsin, which didn't seem to make any difference, so I don't think it's due to the disruption of adhesion proteins as a result of overtrypsinization. Sorry it's a bit different topic, but if you could help me with finding what my problem might be, that would be much appreciated!

If you have mycoplasma you are best off getting rid of the cells; mycoplasmas do all sorts of funny things to gene expression, morphology, etc. It could well be that the mycoplasma are causing the cells to behave oddly as I don't see anything wrong with your protocols there.

bob1 on Wed Dec 1 22:32:44 2010 said:

I discarded all of them as soon as the results came out, and cleaned/autoclaved incubator shelves...

Thanks for your replies, Bob!