Hello Research Gate community,
we are currently trying to differentiate SH-SY5Y cells into dopaminergic neurons. However, every protocol we tried so far did not work out. We tried to induce directed differentiation using RA for 5 days following supplementation with TPA for 5 days in various concentrations (as recommended in several papers). We tried differentiating using RA for 5 days followed by 5 days of BDNF treatment, which also did not work out. Are there more things we have to keep in mind as for example low glucose media or supplementation with Glutamine? We usually use standard DMEM with FCS and Pen/Strep. We would be very happy for any advice.
Thanks a lot for your suggestions.
Hello Britta,
we might have 2 Protocols for you, which you can try.
One is quite complicated, but the other is very simple but more undirected.
We already differentiated dopaminergic neurns with them successfully, but with hipsc.
Depending on your application we might be able to help out.
Contact me via mail anytime and we can see what we can do.
Best,
Stefan
Check up this paper. They also use TPA.
Article Neuroprotection of Indole-Derivative Compound NC001-8 by the...
Hi Britta
In my previous lab, we differentiate these SH-SY5Y cells into neurons using RA+BDNF and we published a paper too. Please see below. Moreover, I want to mention please don't use or Less FBS/FCS if you are differentiating cells.
Article Differentiation Induces Dramatic Changes in miRNA Profile, W...
Hi Tanisha Singh
The method section of differentiating SH-SY5Y is not clearly written in your shared paper.
Firstly, it is not clear with how many cells/cm2 was the experiment started. By medium and the reference stated, I can assume that EMEM:F12 (1:1) with 10%FBS, 1%P/S was used even for differentiation. It is not clear if serum was used for 5 days with RA, however it is clear that with BDNF for 3 days, serum-free medium was used. Blots look good, but immunostaining was not performed to detect TH. Also, how many cells were TH+ and what is the percentage of TH+ cells? Do you detect any other dopaminergic markers? These points are not addressed. It's great that differentiated cells are positive for synaptic markers, but immunostaining is still missing.
It will very helpful if you can address these, as you have shared your paper. Without clear details of any methodology, it is difficult to replicate any result. Thanks!
Hi Anupam Raina
Firstly, you need to plate your cells as per your experiments, be careful about the density of the cells you need to differentiate. If you plate much cells they won't grow neurites or processes and they will die if you plate too less!! You should plate cells so that they can communicate! Secondly, for your ref, plate 100,000 cells in one well of 6 well dish in complete medium with 10%FBS!! Next day, add RA in Medium with less serum (1-5%) and it works really well. Change the medium every alternate day and be careful about RA, because retinoic acid is very sensitive to light exposure and after 5 days add BDNF along with RA in medium with no serum!! Thirdly, we didn’t see TH levels, but other papers have shown good images of TH in Blots and Icc
hope it works for you
Indeed, SHSY-5Y cell line is a dopaminergic differentiated cell line. However, differentiating them into typical dopaminergic neurons needs reprogramming. In that case, using stem cells is more logic.