No, I have not tried N-2 supplement but have tried NS-21.  All the cells in culture dies (can,t distinguish at that stage between glia and neuron)

Hi Sushma, 

Did you use another lot of trypsin? The specific activity often changes from lot to lot. Over- or under-trypsination before trituration can explain increased cell death the day after the culture preparation. The culture medium should not cause the problem because your cultures survived in the same medium some time ago, right? Check anything else that might have changed in due course, i.e. at the time when your culture preparations started to die, including cover slips, the Poly-L-Ornithine etc. By the way, if coating does not work properly and soluble Poly-L-Ornithine persists at the time of plating, cells will also die because the Poly-L-Ornithine is a poly-cation which is taken up by cells, leading to lysosomal pH collapse, similarly to the action of many transfection reagents... Therefore, we normally discard the coating solution and completely dry the coated coverslips before plating... 

Hope this helps. 

Best, 

Jochen

Hi Jochen,

yah I have tried different lot of trypsin.  yah I also got the feeling that media is not a problem.  After PO coating, I wash the coverslips 3 times and then completely dry them . Also have made fresh PO.  previously cultures used to survive for 2 weeks (good enough to do my experiments. But problem started 3 months back and I have tried changing everything but was not helpful. when I increase cell density to almost 3 times, 50% cells survive but then 50% dead cells also there..

Hi Sushma,

Well, in my lab, the primary neuron cultures survive up to 3 months. There might be a general problem with the culture prep procedure so that a minor change would already suffice to have such an enormous effect on survival. We do not change the culture medium and do normally not add AraC or similar. A critical issue may be that the duration of the culture prep is too long. The brains should be removed very quickly (5 min or so) and kept in PBS on ice while isolating the cortices or hippocampi. Did you upscale and need more time? Does someone else do the culture prep? Some other ideas: is the incubator working correctly (CO2) - which color does the medium have? Did you check temperature insight the incubator?

Best,

Jochen

... by the way, are you seeding 30-100K cells in 24 well plates?

Hey Jochen,

Are you guys doing mouse neuronal culture??? I know that rat neurons survive for quite long time but didn,t have any idea whether mouse neurons also survive for that long. I don,t change media and don,t add AraC. I also perfrom dissection quickly but it takes usually 10 min for me to completely isolate cortices. I use cooled PBS.

In my lab, I am the only one doing primary cultures, others are using cell lines.  and I believe that incubator is working perfectly fine because others are also using the same. Still I shifted one of culture plate to other incubator, just to be sure.

But didn,t change anything

Thanks

Sushma

no difference between rat or mouse cultures. Do you treat the glass cover slips? Did you change the mouse company where you order the pregnant mice? 

I am not sure what do you mean by treating cover slips?? But I wash them with 25% HCL followed by 4 ethanol wash and finally bake them. No I didn,t change the company but did change the day of breeding

don't wash the coverslips, just bake them 

Preparation at E17 might also help as neuronal survival is better. I hope that any of my suggestions help you improving the cultures. Good luck! 

Hey Jochen,

Thanks a lot for your suggestions. I will try both the things..

Hi Sushma,

the first days after preparation are the most critical ones using primary neurons.

In generall I would use Accumax instead of Trypsin when working with Neurons.

As Medium I would prefer the Neurobasalmedium of Invitrogen added with BSA, N2-Supplement and ß-ME as well as a Apoptosis-Inhibitor (Y-27632) and GDNF for the first day.

For the coating I prefer Poly-L-Ornithin + Laminin.

Hopefully it helps.

Best Lukas

Hi Lucas,

Thanks for suggestions.

I use Neurobasal medium but with B27 or NS 21, Penicillin streptomycin and glutamax.  I want pure neuronal culture and serum helps in glial proliferation.

can you tell me how ß-ME can help in cell survival?

I want to finally study changes in synapse activity. I am hoping that this does affect the synapse in any way??

And regarding coating, I tried Poly-L-Ornithin + Laminin and compared with only Poly-L-Ornithin. But didn,t find any difference in cell survival.

Thank a ton

Sushma

Hey Jochen,

I am using 6 well plates and 3 coverslips (15mm) per well..

Sorry, BSA was wrong written, also ment B27. Do you use B27 with retinoic acid?

As far as I know we use ß-ME to reduce cystine to cysteine that is necessary for cell proliferation.

Regarding to the coating we observed a better neuronal outgrowth on PDL compared to PLL and best one with PDL+Laminin.

I think the most important step is a precise and fast preparation of the neurons and chilled media till they are in culture.

Best Lukas

Ok. I use B27 with retinoic acid. 

Yah agreed that preparation should be fast and use ice cold solutions.

That,s exactly what I am doing. my cultures used to survive well. But suddenly few months back, they stopped surviving and then kind a become irregular..:(.

Mostly I get one mouse on every friday. I will try all the things which you guys suggested and then will let you know

What conc. of ß-ME, do you guys use.

Thanks

Sushma

Hi Sushma,

we use the ß-ME with an endconcentration in the medium of 50µM. It is toxic in higher concentrations!

Hi everybody,

if everything was working fine (how long?) and suddenly you have neuronal death in one day, could it be because of a contamination of the incubator? Mycoplasmas cannot be detected by visual inspection using a normal light microscope. I suggest to clean and steralize everything and try again with the protocol that was working well. Otherwise I used to prepare cortical neurons from E14-E15 embryos and their were fine, maybe it can help

Regards

Massimo

Hey Lucas,

Thanks a ton.

Susma

Hi Massimo,

I have been doing these cultures from past 8 months and tills 5 months, things were fine. Before me, my lab colleagues had been doing these culture for almost 4-5 years.

I will sterilize the incubator this time before using. Thanks for suggestion.

Best Regards

Sushma

Hi Sushma,

you can also use gentamycin in your medium in the future,  that kills micoplasmaBest wishesMassimo

Thanks,  Massimo. I will try

Best

Sushma

@ Carlos: We actually use horse serum to get rid of neurons, e.g. if we do neuron-"free" glia cell cultures. This works particularly well if we seed the cells at low density and do frequent change of medium. Better use FCS at 1%...

Best, 

Jochen

Hi Carlos, thanks for the suggestions.

 I will go through the paper. Instead of horse serum, I use FCS (10%) in BME medium for seeding the cells and keep them 1-2 hours before changing to Neurobasal with Glutamax.

Best Regards

Sushma

I had similar issues for a while. In my case, the tip diameter that I used for dissociating the cells turned out to be the problem. I started out by using broad diameter Pasteur pipette tips and fire polished them to get tips of reducing diameter for better dissociation. But over the course of few months I kept reducing the diameter too much to get better dissociation. As it turned out, too little of a diameter was shearing the cells and gave unhealthy cultures.

Also, do you use mouse FGF2? They help the cells to sprout out and survive. I coat my slides with poly-lysine and have to wash it several times as residual poly-lysine is toxic to cells. I don't know much about poly-ornithine but I am assuming it could be the same.

I had culture of hippocampal neurons, the cells adhering to poly-L-lysine and cultured in Neurobasal indicated for fetuses (given that I cultivated cells taken from fetuses of Wistar rats).

The medium was further supplemented with L-Glutamine and glutamate, and B27.

When I had the problem of cell death in culture, changed the lot of B27 supplement and the cells were again grown as before. The B27 is rapidly degraded, undergoes the action of light and is essential for maintenance of neurons in culture.

I have helped and I am available.

Thanks everyone for your valuable suggestions..

now neurons in culture looks fine (4 days)

some dead cells are still there but I guess this much you can,t avoid (At least they are not dying just after one day)

Lets see how it goes.

Thanks a lot again

Best

Sushma

Shrinivasan, thanks for the suggestions. I have tried different coatings, but didn,t see much difference. I don,t use FGF2. Regarding dissociation step, I first pre-dissociate with 1ml tip and then go for smaller 200ul tip.

Eduardo, thanks.

one of the first thing I tried was changing B27 lot

Hi Sushma,

If the problem wasn't solved plate some of the neurons in wells without glass coverslips. If neuronal survival improves, order glass coverslips from Assistent (http://www.hecht-assistent.de/en/product-field/microscopy-staining/cover-glasses-for-microscopy.html , Product No. 1001/13). These are the best! Again, don't wash them (as this may reveal toxic glass components, depending on the way they are manufactured. Regarding trituration, we also use 1 ml blue tip for the first two steps, and then 200 µl yellow tip for teh last step. Use of fire polished glass pasteur pipettes may result in varying results...

Best,

Jochen

hey Jochen,

I have earlier tried plating on plastic well, but it didn,t give me much difference result    ( little bit better survival as neurons attach better on plastic)

I assume that the reason of drastic death just after one day was the oxidative stress. I notices that while doing trituration, unknowing I was creating few bubbles (still not sure, if this is the main reason. Lets wait and see how long this preparation survives and if I could repeatedly get healthy neurons).

Regards

Sushma

Please let me know if this was teh reason for increased cell death. Crossing fingers!

Thanks all of you for the wonderful suggestions.

My work is back on Track again..:)

Hi Jochen,  I believe that reason of cell death was indeed the oxidative stress.

Best Regards

Sushma

Hi Sushma,

This is great!

Kind regards,

Jochen

I in cultures performed in hippocampal neurons, I used poly-L-lysine to promote cell adhesion and sowed 20k cells per well in 96 well plates.

In neurobasal medium added B27, L-glutamine and glutamate.

When I was training bleebs, solved by replacing the B27. This is photosensitive and is easy degradation at 37 ° C

check the calibration of the CO2 incubator.

Hi Eduardo,

Now my cultures are surviving well.

Thanks for your suggestion. I will keep a check on these things also.

Best Regards

Sushma

I have had the same problems as you are having and have found solutions to many of them. I use either PDL (100ug/ml) or PEI (0.1%) coating.  You should consider preparing the neurons with an E15-16 as you will get better survival.  Speed is very important when doing the dissections and keep everything on ice.  I dissect the cortices in Neurobasal +10%FBS which improves survival.  Dissociation should be as gentle as possible i.e. Accutase for 20 min & fire polished pasteur pipets.  I plate them in COLD Neurobasal + B27  with Gentamicin and let them warm up in the incubator.  Check your incubator for the highest possible humidity and one that is not opened frequently as neurons are very sensitive to osmotic shock due to evaporation of the media.  I can extend their life span by adding the water that evaporated.

Hello

I am thankful for the queston and suggestions,, I did neuron for the first time and got same death problem,,

I used Trypsin for dissociation,, for plating Neurobasal +B27 +1%pen-strep +1%antifungal,, 

-

I observed the following

- clumps of neurospheres  in giant cells

-Cells started to detach after 4 days and made rolling clumps visible by eye in medium.

-----

My questions please:

-are neurons sensitive for pen/sterp and antifungal.

-what is the best method for dissociation(trypsin, etc.. time & conc)

-how to check purity of culture,, which tech and Markers.

-How to check maturity(time - shape & lenght of dendrites)

-for protein extraction which kind of buffer better to use.

-for immunostaining,, is it safe to do normal washing and fixation with PFA.

-how many time I can leave the cells without  becoming old.

-is it possible to do replating in case of clumping of cells,, and how? by trypsin or scraping..?

thanks

Your dissociation with Trypsin is a little too hard on the neurons.  Try Accutase or Papain.  I would omit the antifungal and use Gentamycin instead of Pen-Strep as it has a broader spectrum but is more gentle for the neurons.  I believe that the major cause of your problem is attachment.  If the neurons do not attach, they either form neurospheres or they die very quickly within the first few day depending on the level of attachment.  Has your supplier of poly-L-ornithine changed?  Try the poly-D-lysine from either Sigma or Millipore if it is available in your area.  I cannot help you with your other questions as I do not perform any of the above mentioned techniques.  Neurons have been known to survive for months but it is very difficult.  If you can maintain them for 10 days and do experiments after this 10 day period, you are mostly working with mature neurons although some papers say that 14 days are required for full maturity.  It is normal to get a constant low level of cell death while in culture as this is what would happen in the brain through apoptosis. 

Yes you can dissociate and replate the neurospheres but not the neurons that have adhered to the plate.  I suggest dissociating the neurospheres mechanically by pipetting without any enzyme.