SHSY5Y differentiation - (Nov/14/2006 )
I have problem with my SHSY5Y cells. I am working with neuronal diferentiation pathways and I want to differentiate neuroblastomas. I tried to use RA 10 uM with serum for one week and then 10 uM RA with 50 ng/ml BDNF for an additional week. But at the end I could not see any neurites. Surprisingly, my control cells without RA and BDNF were having more neurites than the others. Do you have any idea with other GFs like IGF-1, NGF or GDNF that I can try or with the possible problems related to RA and BDNF.
(1) what's your control cells? Please don't tell me that you had mistaken our control as differentiating SH-sy5y (I made this kind of mistake once during the procedures^_^);
(2)Our group tried to differentiate SH-sy5y cells with BDNF to study the expression of NR2B receptors several years ago. That project went smoothly, it appears to me that if you have the right SH cells (in good condition, of cause) and the right prepared BDNF, you can get it done anyway. If I well you, I would check this out first, and then optimize the concentration and time (even BDNF is not cheap);
I'm so sorry I can't remember the detailed information even I did exactly the same thing. But anyway, good luck buddy, you are gonna get it done!
Thank you Darren for your interest,
My control cells were SHSY5Y in +serum medium for one week and then in serum free medium (1%FBS) I did not add any RA or BDNF to these cells. But I saw more neurite extended cells. In one paper I read that IGF-1 also works. Now I will try again with BDNF and also IGF-1. I was working with PC12 for 2 years, I did not have any problem. They were responding to NGF very well. But neuroblastomas are little bit hard to deal. I started to understand. Anyway, thank you again.
Hello all, I wanted to star a topic but it seems to me this is already on the subject.
I am close to start working with SH-SY5Y in a similar way to you, Ozlem, and I would like very much to know if you solved the problem. My first goal is to reproduce a paper (J.Neurochem. 75:991-1003) where with Retinoic Acid 10 µM/ml and BDNF 50 ng/ml (exactly as you) they manage to induce axon-like neurites even 400 µm long. Were you able to see something like?
Thank you and congratulations for this forum!
With retinoic acid 10uM/ml, and a growth factor ( we use neurturin), its possible to get long processes. Also having a good substrate helps to promote processes outgrowth. We do it routinely.
Finally I could solve my problem with differentiation. I did not use RA and BDNF since I have failed. I started to differentiate neuroblastomas with 10 nM IGF-1.
I seed the cells onto collagen coated dishes in growth medium. Next day, after cells are attached, I change the medium to 1% serum medium for overnight and then I add IGF-1 for 2 days. I am sending you my light-microscope images of differentiated and undifferentiated cells. They dont look so bad :-))
Hope you will get nice results with your experiments....
We change media 3 hrs after seeding them with NeuroBasal media.
How old are the cells that ones who have attached. None of them seem to have differentiated yet.
We see full differentiation after 2 weeks of them in media with retinoic acid and neurturin. Ofcourse, one will see differnces after a week. But in 2 days, I doubt it.
What is a "good substrate" for you? I have read some papers where a 0.05 mg/ml collagen is used, in others it is poly-L-lysine. In our lab the routinary coating for dorsal rooth ganglia (DRG) cells is a 0.4 mg/ml collagen solution.
In your pictures, Ozlem, I must confess I am unable to find any differences in terms of neurites extension. It may be just my inexperience in the subject, but besides a more round cytoplasm I don't see any sign of differenciation. As far as I remember, only extensions longer than 100 µm can be called neurites (Oe, 2005). I have also read that, as Scolix says, we must wait at least for one week to see something. Maybe in your cultures it is just too early. In my case I need axon-like extensions as long as possible.
After comparing bibliography I am planning to use a sequential differenciation with retinoic acid (5 days) in 15% FCS, then BDNF (7 days) in serum-free medium and finally a combination of NGF and BDNF in N2-supplemented Neurobasal medium. Does it sound "too much"? I want to be rid of serum as soon as possible to avoid the proliferation of the S-subtype of cells (Encinas, 2000). Do any of you know if N2+neurobasal is still too good for this anoying subclone?
I hope I get the trophic factors next week and I can also add some facts, not just questions, to the debate Thanks for your time!
A good substrate is any thing whihc allows good neurite outgowth. Could be laminin, collagen or even matrigel (which we use).
In order to have the cells differentiated, I would always have the cells in retinoic acid.
We change media 3 hrs after plating to neurobasal with growth factor + retinoic acid.
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...