So, to continue the story and add new questions... (I could've open a new topic, but I feel it is more informative to have all these SHSy5y-related subjects together, if this is not forum's politics, please tell me)

Right now I have already began to differenciate my SH-SY5Y cells with 10 µM retinoic acid (still with serum and no growth factor). After 2 days they are already reshaping and some show nice dendrite extensions. Substrate is 0.4 mg/ml Collagen-I treated with acetic acid. In three days I will change to a serum-free medium plus BDNF for further diferenciation.

However, although I seeded the cells with the density described in most papers (10⁴ cells/cm²), I have the feeling my wells are really crowded, specially in the middle, and I am affraid it may impede longer neurite extensions. Does any of you have any idea of which is the lower density SH-SY5Y can bare with? As far as I understand, if these cells differenciate they cannot divide themselves, can they?

In the meantime I will try a 10³ cells/cm² density...

So, I tried 10³ cells/cm² and, effectively, it was too few. You are right, scolix, that the cells tend to concentrate in the middle; I will use your "90°-tilting system".

By the way, why the bioforum does not advise me when a new post has been added to my last one??

I'll keep this one updated

My cells are differenciating!

I seeded them at a density of 0.5x10⁴ cells/cm² and, after titling, they are evenly distributed. They react with Retinoic Acid and extend neurites...

But my question is the following one. I know that in this cell line there are different cell subtypes, and I would like to know if any of you have experience in morphologically distinguishing the neuronal-like type (N-type). It is just to know roughtly, and before doing any further process (I want to do a co-culture), how many N-type cells I have.

Here I took pictures of the three subtypes I have been able to distinguish. They are from the previous experiment, when I was absolute naif in cell culturing, so don't pay too much attention to the uneven cell density, or the collagen debris I have everywhere.

The first one is this pyramidal one in the middle-right side of the snap, the second these two stellar-like, and the third one the bipolar one.



As far as I understand, the stellar-like are S-type cells, so more epithelial ones, but between the pyramidal and the bipolar one I am confused. I read somewhere that differenciated SH-SY5Y cells are bipolar, but the pyramidal one looks so healthy and so "neuronal" that I doubt. And it is not a single cell like that, there are many.

Also, I would like to know what do you think about the bulbs at the neurite of the bipolar one. Is it normal?

Thank you very much! You cannot know how valuable is all the things you pals are teaching me!

Hi radar did you find out what your different cell types were? i have just revived some cells from liquid N and they all appear to be S-type! they dont look very good at all, i have never seen these cells like this. They are p18 new cells from the ECACC.

help!

thanks

Hello!

I'm trying to establish the cultivation of SH-SY5Y in our lab. The person who did the cultivation beforehand recommended me the following:

DMEM medium with 4,5 g/l glucose with 0,11 g/l sodium pyruvat supplemented with 2 mM l-glutamine, 10% FCS and 1% pen strep.

I use culture flasks and plates von Falcon and Biochrome.

I bought cells from CLS with the passage number 34, but now I'm also hatching a few cells from the passage number 11.

My problems:

The cells from CLS (the other are still not enough for passage) don't get adherent enough in my 96Well plates so that I can't change the medium without removing cells from the plates.

The cells can be removed with t0,025% trypsin/EDTA in half a minute at room temperature.

I have to seed them at densities of 80 000 / cm² otherwise the react to medium change with building "over-easy formation" (http://en.wikipedia.org/wiki/SHSY5Y).

Do you have some ideas for me? What can I change or is it normal that the cells are so lightly attached?

Thank you in advance, Ranuncula.