Hi
I seeded 96 well plates with 10,000 PC12 cells. I did this procedure 3 months ago using the same batch of 96 well plates (expiry only in 2017) and had seen cells growing OK in the wells - they were well spread out and most were not in contact with neighbouring cells. But this time, the day after seeding, the cells appear as in the image labelled day 1. These are taken with 4X objective so you are seeing pretty much the entire well. The cells appear to be stringed with other cells in long lines. The next day they appear as in the image 'day 2'. The cells seems to have conglomerated into a large blob. Have anyone seen this before? What do you think happened?
I think in mycoplasma contamination, you can try to make a test and if it is thus you can try an appropriate antibiotic.
I'm sorry for my english...
Unwanted harmful contamination, is always a serious threat and possibility.
Looking at your cells under the microscope, and recording or photographing the Image(s), is extremely important, and useful.
Always carefully document exactly what you are doing, and working with, for each experiment.
Nice work !!!
Hi William,
You probably have a mycoplasm contamination. I would recommend you to test contamination by PCR. However, if you have stock it is better to discard these cells. Test new stock vials for mycoplasm before entering them into experimente.
Good luck.
Dear William,
While contamination of your stock is always a good thing to check, there are also some other possibilities. In the first photo, it almost looks like the PC12 cells are putting out neurites. This is something they usually only do if exposed to NGF. Were these "primed" cells (exposed to NGF for several days and then either frozen or replated)? The reason I ask is that if they had been exposed to NGF previously, but their media doesn't have NGF presently, you are perhaps seeing NGF withdrawal. I also checked Chapter 18 in Methods in Enzymology Vol 147 (1987) that covers the care and use of PC12 cells, and they mention that exposure to NGF can enhance cell clumping (photo 2?) but that it can be minimized by adding horse serum to the media. Are you using a new lot of horse serum?
Alternatively, you may be seeing the clumping that the cells can exhibit when they don't like the substrate. Unfortunately, I can't help much with your choice of substrate. I have never grown my PC12 cells on uncoated plastic--I have always grown them on air-dried rat tail collagen, or briefly on poly-D-lysine coated glass coverslips for immunohistochemistry. Hopefully someone here on RG has more experience with culturing them on TC plastic.
Good Luck!
Jill
Hi all
Thanks for all the helpful feedback. I have discarded this batch of cells. But is this kind of stringing/blob appearance a specific symptom of mycoplasma contamination?
I've thawed a new batch and its growing well, even in 96 well plates, the same lot I used previously. So I think its not a substrate problem. As for cells which had the problem, they were PC12 cells adapted to low serum (1.5% horse, 0.4% FBS). They had never been exposed to NGF.
Jill that's an interesting point about clumping due to NGF, and prevention by horse serum. So far a lot of the published protocols I read that involved adding NGF to PC12 cells seemed to do so when there is either low or no serum. I presume that is to maximise the effect of NGF. But its intriguing that horse and not other sera has that effect.
William
Hi there I am working with PC12 cells and I have to admit your first image definately looks like these cells are undergoing differentiation. Normally the PC12 cells (unlike say 3T3) are very rounded in appearance even when adhered to surface via collagen. For differentiation, for lots of other neuronal cell types you can use serum starvation to activate neuronal differentiation. In PC12 cells you use media with no serum. NGF presence in media not only induces differentiation but also stops cells from dying.
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