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RA-differentiation of SH-SY5Y

SY5Y retinoic acid differentiation RA

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#1 JHN

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Posted 10 February 2012 - 01:13 AM

Hi,

thank you for this very helpful forum!

I am new to the differentiation treatment of cells and I am experiencing difficulties in achieving differentiation of cells. Any help is very much appreciated.

I want to differentiate SH-SY5Y cells (bought from ECACC via Sigma) with retinoic acid (R2625, Sigma Aldrich). For what I have seen in the literature, differentation should be achieved by treatment of cells with 10µM RA in full medium (DMEM, high glucose, L-Glutamine, non-essentail amino acids, 10% FCS or DMEM/F-12 with the same additives) for some time (3-7 days) with periodic media exchange every 2-3 days.

To my understanding, differentiated cells should stop proliferating and in case of RA on SH-SY5Y the cells should also show neurites. However, my cells simple continue to proliferate (just a bit slower than cells that only see DMSO but still fast) and I cannot see any difference in shape between vehicle-treated (DMSO) and RA-treated cells. I also tried higher RA-concentrations up to 100µM without success.

I solubilised RA in DMSO at 15mg/ml, sterile filtered it and store it at -80°C. When I add it to the medium I see that it immediately forms insoluble crystals that slowly sink to the bottom. I then vortex the mixture until I cannot see any crystals anymore.

Do you use FCS (FBS) in the differentiating media? If yes, how much? I started to think that maybe the growth-promoting effect of the FCS counteracts the RA although I have read that others use up to 15% FCS.

How do you prepare your RA? What do you do about the crystal formation?

Again, help is very much appreciated!

Thank you in advance
Johannes

#2 scolix

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Posted 10 February 2012 - 11:09 AM

I always used to add antimitotics while differentiating these cells. Also RA after a while storing at -80C can degrade, so i get new ones frequently.

We use Neurobasal with B27 as the differentiating media and no FCS.
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#3 JHN

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Posted 13 February 2012 - 12:04 AM

Hi scolix,

thank you very much for your reply! I certainly will try this. Do you also observe that RA instantly desolubilises when added into the medium?

Best regards,
Johannes

#4 scolix

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Posted 13 February 2012 - 11:01 AM

you have to vortex the media after adding RA and it dissolves readily.

#5 JHN

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Posted 15 February 2012 - 11:24 AM

Thank you scolix. I will try this as soon I have all reagents in place. Do you have a special anti-mitotic to recommend? We have Ara-C (Cytarabin) in our lab.

Johannes

#6 scolix

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Posted 15 February 2012 - 11:52 AM

Nothing special about antimitotics. Try it and see how the cells behave, you may alter it accordingly. Also dont forget to add neurotrophic factors. We added neurturin. Many others use GDNF or NGF.





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