Good afternoon,
I have some question related to the handling method for neuroblastoma cells SH-SY5Y.
First, I grew the cells with EMEM medium (10% FBS and 1% Penicillin/Streptomycin). The cells grew well and started to divide. However, the cells aggregated and formed clumps, and the 48H later most of the cells detached from the bottom of the plate and floated in the medium. Since the cells did not attach to the plate, we found it hard to handle and do experiment with the cells.
Therefore, we tried another medium, which is DMEM( 1,000 mg/L- Glutamine, High glucose 4.5g/L, 110 mg/L sodium pyruvate) + 10% FBS and 1% Penicillin/Streptomycin. The cells also grew well at first, but we faced another new problem. The cells did not form many clumps like the first method, but the shape of the cells were changed and some of them still detached from the plate and floated.
(Note, I am aware that there are 2 forms of this cells- adherent and floating forms. But i was told that the floating one is not good to be used in experiment and also I need the attached cells for my experiments)
Hence, I am looking for your recommendations here. Any opinions from all of you are highly appreciated! Thank you!
You can try with RPMI containing 10% FCS. In my previous lab we worked with this cell line with these medium conditions.
Good luck
We have been using this cell line for many years without problems. It's a subclone of SK-N-SH cell line which is a mixed population. SY5Y cells look quite neuronal, not very sticky to plates, and do not often form clumps. We simply grow them in regular DMEM with 10% FBS without antibiotics (antibiotics shouldn't be a problem). You may check your plates to make sure they are for cell culture. Using wrong plates happens at very low chance. But, I did misuse bacteria dishes (which shouldn't be placed in cell culture room) for cells and found cells were very stressed several days after plated. Another thing is that you may check your cells for mycoplasma contamination. If you are not sure the cells came from a clean source, you may get another vial from different stocks. Otherwise, this cell line is pretty easy to handle.
All the best
Cheng
I also grow SY5Y in DMEM as with cheng above. sometimes it takes a week or two for the cells to adapt to the FCS. I would follow the recommendations of the company or person who froze down the cells initially, after you have good freeze downs, change to RPMI or DMEM (10% FCS), what ever works best for your work and check they are growing well. I have seen papers where they use both those media for these cells. Microplasma is one thing to check. Also make sure your glutamine has not gone off, you have to add this and neuroblastoma uses it a bit faster than other cell types. L-glutamine sometimes goes off in a week. glutamax is another option. Not all media has this added already and usually you have to add some fresh on top- I added some fresh on top of what was in the media when I was using L-glutamine.
Olaia Martínez-Iglesias I have never used FCS before. Would you mind telling me the differences between FCS and FBS. I did check for some information about it, but it said there is no big differences between these two serums.
Cheng Xue Thanks for your recommendation. Do you think that the antibiotic is the cause of the problem? I read some papers about SY5Y cells research and most people used the same antibiotics as I do (Penicillin and Streptomycin), but I am still curious about the effects of the anitiotics on the cell growth.
Moreover, about Mycoplasma Contamination, could you recommend some ways that I can use to check this kind of contamination?
Jessica Lilian Bell Thanks for your recommendation.
About FCS, could you tell me the difference between FCS and FBS? I have read some information about this, and they said there are no big differences between these two serums. Moreover, It is interesting to see you metion about carefully adding L-glutamine to the cells frequently. Did you mean I need to add few mL of medium with L-glutamine to the cell plate regularly, or in other ways? In addition, about Mycoplasma Contamination, could you recommend some ways that I can use to check this kind of contamination?
FBS and FCS are the same thing. Fetal Bovine Serum, Fetal Calf Serum. You add the extra L-glutamine to your media bottle or aliquot when you add your FCS. You can keep using this media for a week if placed at 4oC, people then normally add another portion of L-glutamine after a week, or before the next use and use and this is good for another week. if you use glutamax instead of the L-glutamine it does not easily go off and the complete culture media (media with all the extras added) is normally good for a month at 4oC. for mycoplasma, you can use a PCR strategy (ie https://bitesizebio.com/23682/homemade-pcr-test-for-mycoplasma-contamination/, or DSMZ website SOP) , or quickly stain your cells with a nuclei dye like DAPI. the mycoplasma nuclei will also be stained. Antibiotics should not be a problem if you use them at the recommended concentration, it is not essential, so you can remove it to check there isn't a mistake there. When thawing cells, don't spin down the cells just add thawed cells straight to a culture flask (I found with SYSY they liked that better then spinning them down. Gently place in a T25 flask (depending on cell number - you can even add to a 6 well plate if you have a low density) with at least 10 mL complete media (DMSO needs to be lower than 1% in the final thawed mix). Change media between 6-18 hours afterwards (get rid of the DMSO once cells are attached). Make sure lid is loose, or has holes in it. I have accidently tightened a lid that was vented, not with filtered holes and I killed cells this way before. You should use 20 % FCS for the first few days, to help the cells recover from the thawing. Please check the plastic plates/flasks that they are suitable for adherent cells, some plastics have different treatments (as per Cheng's comment above), ie for agar, and also people sometimes buy flasks especially for suspension cells - when they want to prevent adherence. Note neuroblastoma cell lines adhere much better in flasks (compared to plates) - I don't know why, but it makes a significant difference especially with NBLS and LAN-5. So try to use a flask until you have enough happy cells. All the best Jess
Jessica Lilian Bell Thanks so much for these detailed information. I will try to handle these cells according your recommendation.
However, I have one more question, you did mention about the cells do not like spinning down, how about the spinning down during subculture? Is there any recommendation? or is it exceptional to the spinning down during thawing the cells?
Just after thawing - they seem to prefer me to add them straight to a flask with media. They adhere fast enough that you can change the media in 6-8 hours. You can also spin them down at 1200 rpm but you will just get a lower survival rate.
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