Posted 28 November 2010 - 11:20 AM
Posted 28 November 2010 - 02:59 PM
Edited by bob1, 28 November 2010 - 02:59 PM.
Posted 29 November 2010 - 10:51 AM
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!
Posted 29 November 2010 - 02:40 PM
Posted 29 November 2010 - 02:56 PM
Sorry, haven't used the cells at all, but those are definitely senescent.
So there is no way to keep passaging them then??
Posted 30 November 2010 - 02:27 PM
Posted 01 December 2010 - 11:09 AM
No, senescence is permanant. What are your culture conditions? What sort of sub-culture ratios are you using?
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!
Edited by sc_queen, 01 December 2010 - 11:10 AM.
Posted 01 December 2010 - 02:32 PM
Posted 01 December 2010 - 07:57 PM
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.
I discarded all of them as soon as the results came out, and cleaned/autoclaved incubator shelves...
Thanks for your replies, Bob!