I am working on PC12 cells since last 4.5 years and used them as a differentiated model by exposing them with NGF .. It worked very well as % of differentiated cells are about to 99 % but nowadays, only 75-80% of cells get differentiate by using NGF (50ng/ml). this time also,I worked on same protocol, i.e. RPMI with 1-5% serum on PLL coated plates with NGF but i am not satisfied with my differentiated cells.. any suggestions?? :):(
Hi Tanisha,
TrkA receptor with bound NGF will be internalized into the cytosol via endocytosis. NGF will be directed to the lysozome to be destroyed and TrkA will be recycled to the cell membrane.
In other words, NGF is consumed over time. If you want to make a long-term treatment, you should change medium with fresh NGF-containing low serum medium every 2 or 3 days. For a 8 day treatment protocol starting at d0, and depending on the concentration you use, you could gently change the medium at d3 and d6, or only once at d4. Moreover, NGF-treated cells will tend to dedifferentiate and even die if NGF-starved, since NGF becomes a strong survival factor once the differentiation pathway is triggered.
Hope it helps :)
Alexis
The choice will be cell-type specific. For example, COLLAGEN-coated wells or coverslips will be the choice for growing osteoblasts/osteocytes, etc. and I have seen better results for stem cells or cancer cells when poly-L-lysine-coated wells or coverslips were used.
I suggest to use laminine which is well adapted to cells from neuronal origin.
Gelatin can be assessed easily and is really cheap. You may give it a try.
Dear Tanisha,
If PLL has been working for you in the past, then I might suspect the serum or the NGF may need to be replaced (unless you have tried that already). However, since you asked, I have always grown PC12 cells on rat-tail collagen, but then, that is how the lab I worked in always did it. We even made our own collagen, since it worked much better than what was available to purchase at the time.
Are you using early passage PC12 cells each time you start up the cultures for induction? Perhaps it is time for new cells?
Good Luck!
Jill
Hello everyone, May I know is there is any requirement of NGF after 8day Differentiation in PC12 cells, actually I have to treat my cells with toxicants for 72 hours after 8day differentiation of PC12 cells and isolate RNA??
Hi Tanisha,
TrkA receptor with bound NGF will be internalized into the cytosol via endocytosis. NGF will be directed to the lysozome to be destroyed and TrkA will be recycled to the cell membrane.
In other words, NGF is consumed over time. If you want to make a long-term treatment, you should change medium with fresh NGF-containing low serum medium every 2 or 3 days. For a 8 day treatment protocol starting at d0, and depending on the concentration you use, you could gently change the medium at d3 and d6, or only once at d4. Moreover, NGF-treated cells will tend to dedifferentiate and even die if NGF-starved, since NGF becomes a strong survival factor once the differentiation pathway is triggered.
Hope it helps :)
Alexis
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