What is the most applicable protocol for SHSY5Y cells differentiation?

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Dina Ahmed

Thank you so much!

Samir Ranjan Panda

Hi Dina Ahmed

There are many available protocols to differentiate SHSY5Y cells. However, the simplest and efficient one according to me is 10uM Retinoic acid in 2% FBS media for 5 days. The neurite differentiation is well and also the SHSY5Y neurons are TH positive post staining.

Thanks

@Samir Ranjan

Thank you very much, that’s pretty straight forward.

So, I will just adjust the culture media to contain 2% FBS and add the retinoic acid,

any other precautions?

That’s super helpful, thanks a lot.

No that should be enough, 2% FBS (reduced serum to slow the cell proliferation) and 10uM Retinoic acid for differentiation. Ideally in 5-6 days of time you'll observe a good amount of neurites which you can stain with Tyrosine Hydroxylase if your study involves Parkinson's or you can stain with other markers as needed.

Thank you so much indeed!

May I ask while cells are within the well plate for differentiation for 6 days, i add the new media every other day which contains RA again, right?

Thank you.

Hi... Yes Dina. Please change the differentiation media every 2 days.

Thanks,

May you kindly propose a seeding density?

Also, can these cells be maintained for longer than the 6 days using diff. media and/or complete media for further experiments or should be used right away?

Many thanks.

I generally split a 90% confluent flask into 1:3 ratio. It is recommended to grow SHSY5Y cells at higher densities.

Yes you can maintain differentiated cells for longer days until the cells are in good condition.

Thanks a lot! So by maintaining the cells, this means changing the media using differentiating media with RA or just complete media as they’ve already differentiated..

thank you so much.

No you have to keep the cells in differentiation media all the time until experimentation.

Thank you, and if the experiment itself constitutes media changing, shall I use differentiated media all way long?

Sorry I can answer that only if i know details of your experiment. Whether you want to check toxicity? Neurite outgrowth reduction or protective effects of drugs against toxins.

Thank you, I want to check drugs’ effects on neurite outgrowth post injury