Hello all
I'm working with SHSY5Y cells to differentiate them to neuronal cells. I'm following the protocol with using RA, BDNF and cAMP for cell differentiation. I tried it different times but it was unsuccessful. Can anyone one help me to do this successfully?
Thank you
Hi Elham
I use 10uM RA in 2% FBS medium for 7 days. The cells differentiate well with neurites formation and it's the simplest protocol compared to using RA, BDNF, cAMP which takes 28-35 days to differentiate.
Thanks,
Thank you dear Samir for your reply
Which media you are using? DMEM or EMEM?
Have you ever used Neurobasal medium instead?
You mean you just add RA to the medium with 2% FBS, right? are you adding Glutamax also?
Thank you
* I use EMEM medium. Yeah can just use your regular medium in which your cells are optimised and grow well. In that medium just add 2% FBS and 10uM RA ( with anti-anti, glutamax and sodium pyruvate as is there in regular growth medium). Change the medium every 2 days and the cells will be differentiated in 7-10 days.
* I have used neurobasal medium along with cAMP, BDNF and RA to differentiate as well, however it's a lengthy procedure and I could not find much difference in neurite growth between both the protocols.
So I differentiate with RA alone, it's easy, cost effective and works well too.
Thanks,
Elham Amini. E.A Differentiation of SH-SY5Y cells should be strictly monitored.
- Start by culturing SH-SY5Y cells in DMEM/F12 media with 15% serum.
- Reduce the serum concentration to 10% before seeding the cells in 12-well plates coated with polyethyleneimine.
- Follow a subculture ratio of 1:4.
- For differentiation, reduce the seeding density to 12,500 cells per square centimeter.
- Seed cells in culture media with 10% serum, at a count of 5 × 10^4 per well on Day 0.
- Replace media every two days until Day 12, reducing serum concentration to 2.5% on Day 2.
- Supplement all media with 10 µM RA starting from Day 2.
- Further reduce serum to 1% on Day 6.
- On Day 12, withdraw serum completely and add B-27, KCl, BNDF, and db-cAMP to the media.
- Refresh media every four days after Day 12
- continue 12 to 20 days more.
Read our recent study: https://www.nature.com/articles/s41598-024-63005-y
Feel free to contact us at any time for further clarification.