I have been growing SH-SY5Y cell line in DMEM/F12 media supplemented with 15% heat-inactivated FBS. For differentiation, I plated 10000 cells per well in 96 well plates and changed media every day, which contained 3% FBS with 10 micromoles RA, and also I have tried with 10% FBS and 10 micromoles RA.
In 3% FBS, I noticed cell death within two days of differentiation, and in 10% FBS after five days the well was almost filled with cells. So what should be the ideal FBS concentration and seeding density for differentiation?
I have attached image of the SH-SY5Y cell line acquired after 5 days of differentiation with 10% FBS and 10 micromoles RA most of the cells look like epithelial
Hello Harish Kumar B
The differentiation protocol is a three phased reprogramming approach, which involves gradual serum deprivation and the addition of retinoic acid during the first two phases and then a transition to an extracellular matrix where the cells are given a neuronal media that lacks serum.
The neuron-like cells become dependent on neurotrophic factors, such as BDNF, for their survival and the epithelial-like cells, which require serum, do not survive. Notably, during this differentiation process, serial splitting via a brief incubation with low strength trypsin allows for the selection of the less adherent neuron-like cells (for the next phase or harvest) and ultimately results in a morphologically pure culture.
I suggest you follow the protocol which I have attached below.
https://static.igem.org/mediawiki/2018/1/1d/T--Tuebingen--DifferentiationProtocol.pdf
Best.
Malcolm Nobre
Thank you for your suggestion, I have gone through the protocol suggested by you, but due to financial constraints, we are not able to use BDNF. Many researchers have reported differentiation using retinoic acid only with 3% and 10% FBS, but I am not able to optimize ideal conditions for differentiation.
Warm regards
Harish Kumar B
Hi Harish Kumar B
Usually, on my experience with differentiation protocol for SH-SY5Y, the ideal FBS concentration is 1% for the best results - especially for more intense morphology changes. I had used in the past 5% FBS, however, the cells seems to change morphology more slowly during the differentiation process. Also, you can differentiate those cells only with R.A for 7 days - but you will have in the end a neuronal phenotype more broad. If you are looking to have a specific neuronal phenotype then definitely you should pick a different protocol.
Another important point is to make sure that your initial population of cells before start differentiation protocol has just a few epithelial-like cells in the samples. This will make a huge difference in the proccess.
Best Regards,
Renata Torres
Thank you, Renata Torres da Costa will try to differentiate as you have suggested.
Hi!!
I plated 60,000 cells in a 24-well plate using normal F12: MEM-alpha media and incubated them for 24 hours. Afterward, I replaced the media with one containing 1% FBS and 10 uM of RA and allowed it to incubate for another 24 hours. Then, I changed the differentiation media to eliminate any remaining undifferentiated cells and continued to replace the media every 48-72 hours for a total of 7 days. This protocol has been working perfectly for me.
Exitos!
Harish Kumar B Coat the culture plates/coverslips with laminin after PDL coating, you will get better differentiated cells.
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