I have been working with PC12 cells for years. Lately, however, I noticed that they start dying after 3 days of differentiation with NGF . My protocol is the following: I plate the cells onto poly L-lysine coated plates, 24 hours later I treat them with 100 ng/ml of NGF in DMEM supplemented with 1% horse serumand I change the medium every 48 hours. I have no idea why they die and I have tried almost everything. Do you have any clues about what might be going on here? Help please!
Hi Lucina, I have been working on PC12 differentiation as well, and I could say that I got high percentage of the differentiation rate, I also plated PC12 cells into the Poly L lysine coated plates next I seeded the cell with low density (20*10^4/2 cm, 6 well plates) after seeding I have been using this strategy. 24H seeding with RPMI medium contained 10% HS, 5%FBS and 1%P/S, 24H starvation contained 0,5% FBS and after starvation I treated cells with NGF in serum-free medium ( 0,5% FBS + 100ng/ml NGF) for 6 days treatment and medium was replaced every 2 day.
I assume that PC12 cells in serum-free condition might probably die, however maybe you can try with RPMI medium as I mentioned above and compare with your DMEM medium
Hi Lucina,
I have experienced a similar phenomena whereby 3-4 days after seeding onto poly-l-ornithine coated wells and differentiating in 50ng/ml NGF, the cells start to detach and die. The protocol had been used as standard in our lab for years with no reported issues and so we had suspected mycoplasma, though testing has returned negative. Did you ever resolve this issue?
Hi Jacinta,
although I am currently not working with PC12 cells anymore, I can tell you that we ended up "solving" the problem by adding more serum to the culture medium. In our hands, despite being under NGF stimulation, we still have a small percentage of cells that continue to proliferate, so, of course, now that they get more food, this becomes a bigger problem. Anyway, this was the only way we were able to stop them from dying after NGF addition. You could also try different cell batches, or if you could get a brand new batch of PC12 cells, like from ATCC, maybe you could start over without these kind of issues. I hope you can work it out !
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