That looks like late passage to me.

What I use: DMEM (High glucose w/ pyruvate), and then I supplement with 10% FBS, 2mM Glutamine, and I do use antibiotics (100x anti-anti from Sigma, contains penicillin, streptomycin and ampotericin).

Also, do not use surface adherents on my stocks of cells, as I grow them on plastic.  However for imaging purposes I grow my cells on glass and as such I use acid washed coverslips that are coated in poly-d-lysine (high-molecular weight version) followed by 50uM laminin.

Attached is a picture of cells I just took, pulled them out of liquid nitrogen a couple of hours ago (sorry its not the best picture, used an old microscope as it is quicker).  You'll notice some cells have neurites, totally normal.  With fresher cell stocks, you will notice more round cells, and as passage number increases the cells take on starfish shapes and start growing neurites at higher rates and of longer lengths.  Morphologically, the neurites will begin to look different too, less straight and more jagged, and cells may begin to produce higher numbers of neurites/cells.

What I'd suggest is starting with a fresh stock pulled from LN2, expand onto multiple plates and make tons of stocks.  Each time you thaw make more stocks.  I tend to throw out plates a lot because after about 8 passages they begin to do exactly as you describe, start growing neurites and changing their phenotypes a bit.  Can make immunostaining and biochem a bit inconsistent as well.

Lastly, I am attaching 2 pictures of an early passage cells differentiated for 2 days in dibutyryl-cAMP, they look a little different from yours, which leads me to believe they are simply old cultures.

Sorry about the long response, let me know what you think,

Eddie

Hi. I was working with a neuronal derived cell line and I've noticed that its morphology depends of matrix you used to growth the cells. For example, in glass the cells were amorphous, while in plastic plates (collagen treated) the cells adopted a fusiform morphology.

I also used a low serum medium (2%), the first week the cells seemed really bad, but after the second week (changing the medium every two days) the cells took a more like-neuronal morphology.

that's my experience

(sorry, my english it's not so good, but the idea is there)

Dear Yvonne

I used the serum deprivation method in N2A cells. After 24hs of deprivation (just DMEM+ antibiotics) you can observe differentiation. Anyway, you can do a time curve around this time, to reach your desired conditions/morphology.

Good luck

Once differentiated, do the cells have any properties intrinsic to a particular class of neuros? e.i. DAminergic or cholinergic?  has anyone classified these changes in terms of proteomics? Sorry to not provide answers, and only present more questions!!

We had shown that RA will induce dendrites in N2a cells with also low serum, 0.5-1%. If you also add 0.25-0.5M dbCAMP you not only get dendrites but axons and what appears to be synaptic connections. You can keep thm that way for at least a week.

Hello Kelly, this is what I've found about N2a and neuron types:

http://www.ncbi.nlm.nih.gov/pubmed/19903493

Hi. I am having more or less the same issue.Morphologycally using DMEM enriched with 2% serum cells diferentiate like neurons 24h after deprivation but they don´t express neither Dopamine, neither Glutamate receptors. And after 48h of deprivation cells look really bad. 

I am working with N2a cells now. For some time , we tried to differentiate N2a cells. Then we just want to culture normal N2a cells to do experiment and the normal N2a cells grew very well for the past 7-8 months. However, from the past two weeks,  the normal cells are differentiating itself with the same media we used for culture the normal N2a cells. First, we thought that there might some problem with FBS, but it did not improve too much with the new FBS. Another thing is that we used the new-bought DMEM from 2 weeks ago. I don't know whether it is the problem.  Did someone working on N2a cells run into this problem? Thank you~

Hi Mengya, I have had precisely the same problem except it was after only 5-6 months. I used the same media recipe and brand of reagents in making up my media so it was nothing new there. I believe it has to do with the cells getting stressed. If cells didn't have their media changed enough or they got to a very high passage number, they would start to exhibit the differentiated type morphology. When my N2a cells got to this point, I started passaging a new line of N2a that were 'younger' (had a lower passage number). 

In my experience the N2a cells don't really look healthy after retinoic acid treatment (0.1 uM) so I started serum starving. I do a gradual serum deprivation going from normal 10% to 5% to 1% to 0.1%. They look much healthier that way. Some cells will even survive for up to 2 weeks or more. I also noticed that the plating density matters, so you might have to optimize that for your plating area. I have attached a picture of a differentiated N2a cell from the method described above. 

Hi Raj,  are you using All trans-Retinoic acid (ATRA) for differentiating the cells or the other forms ? Are you seeding on cover slips (glass) to differentiating these cells?

I have tried using ATRA but my cells didn't look very healthy after that treatment so I simply do gradual serum starvation. The picture above was on a 10cm plate (plastic plasma coated). I have seeded on coverslips coated with (PLL + Laminin) or ECM. 

Miguel, I am also interested in obtaining differentiated N2a cells expressing dopamine receptors.  I was planning to use serum starvation but it doesn't sound like you got good results that way.  Did you ever find a better method to differentiate your cells?

Sorry if this is too little too late but we use a non-hydrolyzable cyclic AMP analogue called dbcAMP to differentiate these cells, for other purposes though.  However, exactly what you are trying to do has been highlighted in this publication, using this differentiation inducer:

http://www.ncbi.nlm.nih.gov/pubmed/19903493

Thanks, Ed.  I will try this to see if I get better dopamine response.

You should try retinoic acid in low serum, see Brain Res. 1986 Feb;390(1):99-109. If you want, also try RA and about 0.5mm db CAMP

We use retinoic acid in the 1% FBS. Sometimes, the RA caused the cell death the first day,so we changed the media with 1% FBS and then after one day, cultured again with RA and 1% FBS. You may observe the change of morphology after 3 days and you can wait for a long time. I do not know whether it can change back in normal media. But When you treat the cells with RA or 1% FBS a, the density of cells should be appropriate, around 40%.

so, correct me if I am wrong... when I thaw the N2a cells and I seed them, change the medium (with 10% FBS) in order to avoid dmso toxicity then cells should multiply but maintaining the round morphology, always? if I don't induce differentiation I mean. If while growing (3rd-4th day after thawing), they start to generate neurites this may mean that they are stressed and/or they are old? Thanks!

you can thaw the cells immediately centrifuge the cells. than take it out the supernatent and add freshmedia (37o c). and plate it on PDL coated or even without coated flask and next day you change the media and wait for 3 days. then change the media with lesser FBS like 1%. and wait for the change in morphology of cells.  we are using without serum media to grow the cells

I am having some difficulty to maintain N2a cells undifferentiated. I am using DMEM supplemented with FBS and L-glutamine and without antibiotics, but within 2 to 3 days all of them show differentiated morphology. I have considered if it was due to the presence of sodium pyruvate, but I dont think that it is the reason. I have even tried to plate the cells in Opti-MEM, but they are differentiating also in this medium! Does anyone have a clue why this might be happening? Do you think I should just thaw a new vial? Many thanks!

Joao, do you have a picture you can upload?  I work with these cells a lot, can tell you there are a couple of factors that lead to spontaneous differentiation.

FBS: Non-heat killed and 10% final concentration is what I use.  Serum starvation can induce differentiation.

What passage number are you on?  I typically throw out my cultures anywhere from 7-10 passages as they begin to slow their growth rate down around that time and begin producing longer neurites spontaneously.  Their doubling time should be ~24 hours.  I've also noticed after continued passage their morphology begins to change

Also, at least for me, these cells if well-adhered will produce neurites without differentiation, but with sustained differentiation you get  very long neurites.  The best inidcator in my experience is growth rate.

And lastly, stressors can cause differentiated morphologies.  An example is the poison Zeocin, which destroys DNA.  Zeocin will cause the cells to produce neurites like crazy before ultimately destroying them.  I am not saying there is Zeocin in your media, but it is possible something is stressing them, like a latent mycoplasma infection.  I'd have them checked, as it is well known mycoplasma can induce odd phenotypes.

Let me know if I answered any of your questions or if you have others.

Best,

Eddie

Edward thank you very much for your answer! I cannot tell you the exact passage number since I got these cells from a colleague of mine that didnt know it. I uploaded a picture of the cells!

What is the medium you use? Is it  DMEM without antibiotics too (with the 10% FBS)? Also, do you use something to cover the surface and help the cells to adhere, such as gelatin? I usually don't have issues regarding adherence so I do not add anything  with that purpose.

Once again, many thanks!

Best,

João

That looks like late passage to me.

What I use: DMEM (High glucose w/ pyruvate), and then I supplement with 10% FBS, 2mM Glutamine, and I do use antibiotics (100x anti-anti from Sigma, contains penicillin, streptomycin and ampotericin).

Also, do not use surface adherents on my stocks of cells, as I grow them on plastic.  However for imaging purposes I grow my cells on glass and as such I use acid washed coverslips that are coated in poly-d-lysine (high-molecular weight version) followed by 50uM laminin.

Attached is a picture of cells I just took, pulled them out of liquid nitrogen a couple of hours ago (sorry its not the best picture, used an old microscope as it is quicker).  You'll notice some cells have neurites, totally normal.  With fresher cell stocks, you will notice more round cells, and as passage number increases the cells take on starfish shapes and start growing neurites at higher rates and of longer lengths.  Morphologically, the neurites will begin to look different too, less straight and more jagged, and cells may begin to produce higher numbers of neurites/cells.

What I'd suggest is starting with a fresh stock pulled from LN2, expand onto multiple plates and make tons of stocks.  Each time you thaw make more stocks.  I tend to throw out plates a lot because after about 8 passages they begin to do exactly as you describe, start growing neurites and changing their phenotypes a bit.  Can make immunostaining and biochem a bit inconsistent as well.

Lastly, I am attaching 2 pictures of an early passage cells differentiated for 2 days in dibutyryl-cAMP, they look a little different from yours, which leads me to believe they are simply old cultures.

Sorry about the long response, let me know what you think,

Eddie

Eddie, once again thank you very much for your help. Your cells, even when differentiated, look a lot different then mine indeed. It is weird in fact that I am using the same medium as you and I cannot maintain the cells undifferentiated. Maybe you are right and the vial that was given to me was at a high passage number, but the person doesn't know the passage number. Do you think it would be possible for you to send me one vial? Here in my lab we have a FedEx account so all the shipping costs would be on us! If it would be fine for you, could you contact me by email ([email protected])?

Thanks!

João

Hi there, I have N2a in culture but my problem is that they do not differentiate and seem to be indifferent to starvation. Now after a week in low serum 1%, they are starting to detach (I have of course changed the medium every other day) but not really differentiating. I got those from a colleague and they are at passage 42... Am I wasting my time and simply should recover new cells? Also I will need to grow them on coverslips eventually. I read about pre-coating the coverslips, is that mandatory for appropriate differentiation? 

Hi,

try using a newer stock. I have never used them past passage 20. I also lowered the serum to 0.1% but supplemented with ITS (Insulin-Transferrin-Selenium) and that seemed to help with the health of the culture. 

The cells "differentiate" better on plastic than on glass coverslips in my experience (even when I coat the coverslip with PDL+Laminin) however, this difference is only morphological (longer, more branched processes)  and I guess you will have to define the end point for what you would call differentiated. For me, I used the pan-axonal (NF) (SMI 312 covance) as a marker for differentiated vs not. 

Hope this helps.

Morgane,

Try using dibutyrylcyclic-AMP for differentiation instead of serum starvation.  We typically only use serum starvation for max a couple of days, detachment is one thing that occurs when the cells die.  Serum starvation for a week unless you supplement will kill the cells.  Also from my experience dbcAMP is a better differentiation method as the neurites and cell bodies look much more canonical of neurons, at least from a morphological perspective.

Also definitely use newer stocks, according to ATCC these cells have highly unstable karyotypes.  As Raj had mentioned, definitely throw the stocks away after repeated passage.  He does 20, I actually throw them away after 10 passages myself, but it all dependent on your hands and your protocol.

And lastly absolutely precoat glass, its rather hydrophobic and adherence is poor.  I use this protocol (the poly-lysine and laminin coating one) :

http://www.sigmaaldrich.com/technical-documents/articles/biofiles/laminin-product-protocols.html

Also 2 other things in this regard.  First, I would ABSOLUTELY recommend acid washing your coverslips first.  I use this protocol (http://labs.bio.unc.edu/Salmon/protocolscoverslippreps.html) minus the sonication, and the difference is drastic.  

Secondly, in relation to poly-D-lysine, make sure to use the high MW version of it (>300,000 Da).  This is standard practice for neural lines.

Let me know if you have any further questions

Thank you so much Ed and Raj! Definitely going to get newer cells going and will try the dbcAMP. Wish me luck!

I have wasted my precious time for culturing and differentiating N2a cell lines for more than a year.  None of the protocol works.  Be clear and precise what you want to do first.  Retinoic acid will never dissolve in media.  It will be soluble in methanol or DMSO.  If you try to dissolve retinoic acid in any of these and dilute with media it will become turbid.  Finally, what you are going to do with differentiated N2a cells-neuron-like morphology and physiology? Check the expression levels of differentiating proteins first and then do other experiments. 

I am keeping n2a into 2% FBS containing medium with 20uM ATRA for 2 days. Morphologically I can see difference between undifferentiated and differentiated cells, but I could not get difference in the expression level of Tuj1 protein(which is one of the widely used marker for differentiation) in western blotting. Does anyone know how to explain this situation?

 Gouri,

Don't consider N2a cells as neural stem cells.  They are neuroblastoma.  They even express neurofilaments in an undifferentiated state, which is something you don't normally see until late in neuro-differeniation.  N2a cells might not be the best choice for this sort of assay, since Tuj1 shows up somewhat early during neuro-differentiation, and these cells are already quite committed.

Also consider your culture conditions.  Most people, including the ATCC, recommend culturing these cells in 10% FBS.  Keep in mind this is a 5-fold difference in FBS concentration:

https://www.atcc.org/products/all/CCL-131.aspx#culturemethod

ATRA and dbcAMP for differentiation produce radically different morphology and gene expression profiles in these cells.  Cytoskeletal differences are of note, which may pertain to Tuj1, as it is a component of the microtubule:

https://www.ncbi.nlm.nih.gov/pubmed/2994850

https://www.ncbi.nlm.nih.gov/pubmed/3512042

I suggest trying dbcAMP.

Also it might not be possible in this cell line.  Perhaps a less committed cell line, such as P19 may be of more use, as they are less committed in regard to differentiation:

https://www.atcc.org/Products/All/CRL-1825.aspx  

Thanks Edward for your reply. Can you suggest any other protein marker apart from Tuj1 which can be used to study neuro2a differentiation? Has anyone tried to study neuro2a differentiation by growing them in 10% FBS containing medium followed by 2%FBS with 20uM RA for 2 days and then 1% FBS containing media with 20uM RA. Since this method involves use of retinoic acid and serum deprival, it might enhance neuronal differentiation

I'm not sure, but from my own experience serum starvation is actually a little toxic to these cells.  However we used to do 0.5% FBS by itself.  I have done 2% on its own and it didn't appear overtly toxic, but I didn't do extended analysis, just a couple of days post-differentiation.

The best marker of differentiation that I know of for these cells is a monoclonal antibody called RT97.  It recognizes a developmentally-delayed phospho-dependent epitope on the C-terminus of neurofilament-heavy, so it has a propensity to label axons.  

Here's one of the original papers from the team that generated this antibody:

http://onlinelibrary.wiley.com/doi/10.1016/0014-5793(82)80160-9/abstract?systemMessage=Pay+Per+View+on+Wiley+Online+Library+will+be+unavailable+on+Saturday+15th+April+from+12%3A00-09%3A00+EDT+for+essential+maintenance.++Apologies+for+the+inconvenience.

And here is a commercially available source for the antibody:

http://www.emdmillipore.com/US/en/product/Anti-Neurofilament-200-kDa-Antibody%2C-clone-RT97,MM_NF-MAB5262

Although I do want to be explicit that I have not worked with that source of the antibody.  All of our RT97 aliquots are dervived from the original hybridomas.  

For my studies, I need to cultivate proliferating and differentiating N2a cells in a 24-well plate on coverslips up to 3 days after transfection (using Lipofectamine 2000). I am currently having trouble inducing differentiation with ATRA ( barely any neurite outgrowth or no significant difference to proliferation condition) and I believe it's due to the cell density I plate before transfection. I heard that the cells differentiate well at 30-50% confluency. However, may transfection rate decreases with a low cell density. Any ideas on how to proceed with this experiment? And, is there a marker that I can use for ICC analysis to test positive differentiation? I was planning on using anti-TAU.

Thanks in advance!

Alex

Growth Media: DMEM, high Glucose, L-Glutamine, 10%FBS, 1%Pen/Strep, 1% 100mM Pyruvate

Differentiation Media: alpha-MEM, L-Glutamine, 0%FBS, 1% Pen/Strep, 5uM ATRA

Cell density per well: 50.000 cells/500uL

I would try increasing the serum concentration to 0.5% and to me the ATRA concentration seems too high. I've used up 100uM.

You can try electroporating before plating the cells.

Attached a picture of cells differentiated by gradual serum (over a week halving the serum everyday) starvation and 10uM ATRA + ITS

@Alexander Jäck

I typically seed my cells at 50,000 cells/200ul in a 8-well ibidi chamber slide for ICC. How many days do you allow your cells to proliferate in growth medium? I usually change to differentiation medium (20 uM Retinoic acid) after 48 hrs. For ICC markers, MAP2 and Synaptophysin usually yield good results. Good luck!

Raj M Rajebhosale thanks, will try to increase the serum %! Yes, of course I meant 5uM :)

@Lucy He: I usually let them grow for 24h, than transfect. After incubation of the transfection reagent I either add growth medium or differentiation medium. Then they should either dif. or prolif. for up to 3 days. At timepoint 1,2 and 3 post transfection, I was planning on doing ICC and Western analysis.

Also, I found Tuj1, Neurofilament, Tau to be suitbale markers of differnetiation. Any experience?

I used RA to induce N2A cell differentiation. And I used Tuj1 and NeuN to test differentiated cells in ICC analysis. The expression of Tuj1 is higher in RA treated cells than control cells. However, there were no NeuN-postive cells in RA treated cells. I am wondering that whether NeuN can be a maker for RA induced N2A cell differentiation. Thanks a lot!

I am initiating to study the effect of gamma radiation in N2a cells. Any body have any experience in this area?? Kindly share your view..

Thanks

Dear Y Couch. mTOR Plays a very important role in neurobals differentiation. If you need to your N2a cells to differentiate directed to neuron you might need to go though this paper

https://www.sciencedirect.com/science/article/pii/S0898656807003701

Chunhui Ma I used NeuN as a marker for my RA-induced differentiated N2a cells as well and I was able to detect a time-dependent increase from 0 hr to 48hr to 96 hours.

Lucy He Thanks for your response! I tried NeuN again, and I observed that there were more NeuN-positive N2a cells treated with RA than control cellls.

Retinoic acid in 1% serum produces dendrites. If you add 0.5mM d cyclic AMP you will see axons as well.