HI,

Our group had a vast experience with this model. Please read Encinas M (J Neurochem. 2000 Sep;75(3):991-1003.). Usually we had the cells with 15% FBS + the RA (10uM). Don´t forget to coat the plates with collagen. After 5 days (cells stop but not fully differentiated into neurons) we added the BDNF. Few cells at this step may day and only survive those that are more neuron-like (with TrkB expression). However, never forget that this is a Neuroblastoma cell line and some pathways may be not representative of primary neurons. Good luck!

I agree completely. We have used either collagen or polylysine coated plates and also added NGF and BDNF even though the medium contained serum all along.

Thank you very much Dr. Segura and Schor. I will try to do it. Best regards,

Marcelo

the response of these cells without robust differentiation is entirely different from that with every possible effort to differentiate. for example, the undifferentiated cells actually enjoy Abeta!

I can recommend reading S. Pahlman (http://www.ncbi.nlm.nih.gov/pubmed/7576944) for different methods of SH-SY5Y differentiation. I used retinoic acid (10µM - use TrkB as a marker) or TPA (16nM - a phorbolester - use NPY as a marker).

Good luck!

We first showed in Matsumoto et al Cancer Res. 1995 Apr 15;55(8):1798-806.) that retinoic acid combined with BDNF would differentiate SY5Y cells. This will occur with 5uM Retinoic acid + BDNF.

We kept serum at 10% during differentiation. Watch the glutamate concentration of the medium as well

I use 10 uM all-trans retinoic acid in DMEM/F12 1% SFB and it works really well. I change the medium every 2 days and keep the plates in the dark, even inside the incubator, because all-trans retinoic acid is very sensitive to light exposure.

Another thing you have to be careful about is the density of the cells your differentiating. If you use too much cells they won't grow neurites and with too little density they'll die; you have to give them space to outgrow neurites and "communicate" with each other. There's a paper from colleagues in my department that may be of use to you: Brain Research 1337 (2010) 85-94.

Many thanks Dr. Barbara.

I'll try it.

Best regards,

Marcelo

hi, Nina Schor,

I want to know that is there any difference in the coating with collagen or polylysine? which type of polylysine is used in your protocol, PDL or PLL?

sincerely,

Xiangchun

It seems like you lower FBS% = lowered RA concentration. But I use 20 uM RA which is slightly higher with 10% FBS. The cells differentiate nicely. The one thing to watch out for would be to keep the cells in RA for a maximum of 3 days, then change to fresh media.

I differentiate them with 10 uM RA in 1% FCS for 5 days, followed by 2 nM BDNF in serum free media for 2 days. They grow well and don't die. This gives me cells with extended neurites and robust expression of Tau protein.

cells only need to be treated with RA for 2-4 days. after that replace the media with new complete media. also RA oxidizes when exposed to light.

Add BDNF

This is actually not an answer but rather a question: how do you differentiate the cells in 96 well plates? Or do you differentiate them in a larger plates and then after differentiation seed them into 96 wells and perform the assay? If the later is the protocol, how long do I have to wait until the treatment?

I wouldn't try passaging differentiated neuron. It might be possible if you have a very delicate passaging method, but otherwise it will probably mess up your neurites. I've plated cells in the presence of RA and they differentiate just fine. So technically you can treat cells on day 0 of a passage. Just make sure you have an appropriate basement membrane (eg, collagen) otherwise they won't attach.

Thanks for the answer.

How many cells do you plate in a single well (96 plate) to obtaine good density (after differentiation with RA) for later assays? I suppose 5000 cell/well is appropriate?!

Yes, 5k is good. But make sure they aren't clumpy on the plate. Otherwise they wont differentiate well, and they won't be very useful for imaging purposes.

Right now I am also try to differentiate SHSY5Y cells. I also have some question for this. Usually, we use differentiated SHSY5Y cells as AD or PD model. and also when we culture cell line, we add streptomycin to avoid contamination. Dysfunction of mitochondria play an importmant role in neurodegeneration. Streptomycin, theoretically, might dysfunction mammalian mitochondia. So I am thinking during differentiation, or doing treatmen, we need to avoid streptomycin.

Just be careful with contamination. Before doing any assays I'd suggest doing a mycoplasma test and a gram stain, just to make sure there isn't anything else in culture that might be hurting the cells.

Also you could always do a ATP assay to compare mitochondrial function between differentiate sh-sy5y with or without streptomycin in culture. If they aren't different, you could probably save yourself a lot of trouble with potential contamination issues.

Do we add antibiotics to the media/cells during differentiation?

including antibiotic should be fine.

Thank you Martin!!

Add BDNF.

xu chao-jin thats just one of their growth characteristics. you can get a better monolayer by pipetting your cells extensively with a p1000 and p200 (approx 100-200 times with each)

Do you use DMSO to dilute the RA?

If use DMSO, use de stock concentrated (1000x), so the DMSO is very toxic for this cell line

Hi

I want to add aquestion:

Im about to start working with this cells and wonderd how to keep the cells after the 4 days RA treatment? is the compleet regular medium is fine? how long they will survive?

thanks in advance

Use OPTI-MEM by Invitrogen

Can I differentiate SH-SY5Y directly after freshly reviving them? Wouldn't I dilute out the dmso when I change the media every 2 days? 

THere is some heterogeneity in SY5Y cells. SOme SY5Y will differentiate in ATRA alone while others require the addition of exogenous BDNF. We first reported the differentiation of SY5Y with ATRA (5uM)  and the addition of BDNF in Matsumoto et al Cancer Res. 1995 The half-life of ATRA is 48hrs in tissue culture, in dark, 37oC with 10% FCS. If your cells are dying, add BDNF. 

Dear Marcelo, Please differentiate without FBS for a robust and better phenotype. Then you can test that model for toxicity. Once differentiated in media containing 10uM RA without FBS , you will start seeing neurite projections. Please refer to our newest article in eNeuro.

can anyone recommend me of a protocol to differentiate these cells into dopamine, norepinephrin and serotonin secreting cells?

thank u!!!!!!

Maya Cohen for a dopaminergic phenotype it is recommended to co-incubate a phorbitol ester along with the ATRA. for the others and more on it, refer to this paper, it has some good info on it. Article Considerations for the Use of SH-SY5Y Neuroblastoma Cells in...

@Katerina Christofides

thank you for your replay

in what concentrations of ATRA and phorbol did you treat the cells ?

did you use complete media? (10%FBS)

when treating them with phorbol, ATRA and complete media they didnt stop proliferating..

did you replace the media while differentiating them to a different type of media?

thank you for your kind replay!!! :)

So, in many protocols it refers to using either a low FBS concetration or no FBS. This is dependent upon your cells,

for me my sh-sy5y's preferred 3% FBS with 10micromole ATRA. The proliferation didnt stop but it did massively slow down.

So when i made up my media, i used the usual DMEM/F12 HAM, 3%FBS, 1%NEAA

added 10micromole ATRA, then added to the cells. I kept checking on them every day, it normally takes 3-5 days. When i did it, it took the whole 5 days to get the whole culture differentiated.

Hi,

Please can anyone tell me if the correct! For 10 days differentiation ( 10uM RA), I dissolved RA in DMSO ( 3.3 mg/ 1 ml DMSO) then I stored RA in -20C for one month. before the differentiation experiment, I RA diluted in ethanol 1:10 then added to the medium at this dilution ( 1:1000) , or some time i added The RA directly to the differentiation medium at this dilution (1:10000).

The differentiation medium that I used ( DMDM/F12- 1%N supplement- 1%Glutamine).

However, the cells were died. I am not sure if there is something wrong?

Thanks

Dear Bader,

I am using all-trans RA as well for differentiation. The RA powder is dissolved 3mg/ml DMSO according to manufacture instruction to have 10 mM concentration (stock solution). For example, if you have 50 mg, it will be dissolved in 16.7 mls DMSO. Then, after complete dissolving, filter it using micro-filter 0.2 um inside the hood and aliquot 0.5ml or even less amount in black eppendorfs (RA is so sensitive to light so it should be kept in protected eppendorfs. The stock should be kept in -20 C.

To prepare your final concentration in the culture media, the DMSO concentration should be not more than 0.01% v/v to keep the cells alive. So, you should dilute your stock in the media directly (not ethanol or more DMSO). To get 10uM concentration, I recommend using the Dilute a stock solution part of this molarity calculator

https://www.graphpad.com/quickcalcs/molarityform.cfm.

I hope this could be helpful.

Regards,

Hi

Please any one help me

how long I should treat with RA before treat my differentiated cell with toxicant? What the standard criteria I should estimate: morphology changes only? I try to do 3 days and 6 days but actually the morphology not change much and in some papers they suggest to use protein biomarker but my lab dont have it yet. So can I just base on the morphology to decide if I should continue treating my cell with toxicant? Should I change RA medium every day? And during the toxicant treatment do I still need to maintain RA treatment in the cell medium? Is that the right thing if I base on the counting cell by trypan blue to decide if the proliferation is decrease or not?@ Thank you so much

Thank you so much for your reply. Abhishek Jha

one more thing which criteria I can base on to sure that I am successful to RA treatment and can continue treat with toxicant?

thank you so much@

@ Abhishek Jha

are these markers for spesific type of neurons? dopamonergic?

Thank you! :)

Did you try to differentiate them to other type of neurons? serotonergic? GABA?

Maybe using another cell line?

Is there a spesific cell line and method you can recommend on?

Abhishek Jha