how long did you coat the cover slip with PLL? After coating did you wash the cover slip with the PBS/HBSS?

Usually I coat the cover slip with PLL overnight, then I washed with PSB or HBSS 3 times. Wait until cover slip dried, and you can start plating. I used concentration 0.5 mg/ml PLL (MW=70-150kDa) or 1 mg/ml PLL (MW=30-70kDa). and don't forget to changed the half of the medium every 3 days. hope it's work.

Or you can read this link and watch the video for primary culture of hippocampal.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872976/

http://www.jove.com/video/895/primary-culture-of-hippocampal-neurons-from-p0-newborn-rats

Dear Vini,it is often the coating that kills the neurons. Sometime Poly-D-Lysine is the better option,  as neurons (and other cells) can release proteases that break poly-L-lysine. So, try that! We coat with Poly-D for ca. 2h. Important, do not fully dry your coverslips (not to 100%) and never for y long time. They should be like 95% dry, but not wet. Good luck!

Hi Vini,

Hope you are doing good !

Your preparation does not seem to be problem since the neurons look healthy after prep. It can either be coating or media. Coating seems to me the prominent reason because looks like after 1-2 days, neruons are retracting their processes.

You can try Poly Ornithine (PO). I use the same and after overnight coating in incubater, wash the coverslips 3-4 times with water. Then dry them in Hood.

You can also try to use new B-27 in media because sometimes  the B27 lot itself is bad.

Also, I have a general question. Is there any specific reason behind using p0-p3 pups? because for neuronal primary neuronal, majority of people uses E17-18 foetus

Best Regards

Sushma

Hi Vini,

I only have a few points to add to previous posts:

- are you using inactivated FBS? for many primary cultures the presence of the complement may be cause of cell death.

- have you check that the temperature/CO2 concentration on your incubator is correct? sometimes looking at the digital screen is not good enough, I would recommend manually test for both parameters.

- as Sushma indicates cultures from rats are normally done at the embryonic stage while the ones form mice are done at postnatal p2-p4; any reason you can not use embryonic cultures?

Hope this helps,

Ezequiel

Thanks, everyone. I will try ornithine. What should be the MW of it, does it matter? I am not using heat inactivated FBS, will do that. Anyone has experience with Neurobasal-A medium? Or can you share your thoughts on the medium composition? Eg. Should you add FBS and B27 together or just B27 itself is enough?

Also, just using P0 rats because I did that for mice and dont have experience with prenatal :-(

Dear Vini,

As to your question regarding media composition, Neurobasal A was formulated to be used with serum-free supplements, either N2 or B27.  Some people add a small amount of heat-inactivated FBS to the media used to plate the neurons, or to inactivate the enzymes used to dissociate the tissue (if they are using that technique).  Then, after the neurons have attached to the substrate, they switch the media to the serum-free Neurobasal A. 

I also use Poly-D-lysine, rather that Poly-L-lysine, as Natalia suggested.  I also make sure that the plates or coverslips are well rinsed before seeding the neurons.

The source of the papain can also be critical to the health of the cells.  Papain from Worthington is tested for the Huettenr & Baughman postnatal cortical neuron protocol in "Culturing Nerve Cells" edited by Banker and Goslin, and I have had good luck with that protocol.

In addition, you may try inhibiting the action of the papain with ovomucoid trypsin inhibitor and BSA, rather than with FBS (the recipe is in the protocol above). 

Good Luck!

Jill

I just gave a go to a culture with ornithine/laminin coating on coverslips and Neurobasal-A (serum-free) as the medium and I have the same results with dead cells :-( I still saw good neurons in the microscope before plating though. 

I am starting to think if the problem this time is the dissociation or the initial plating medium. For example, if I use papain for dissociation but use serum-free neurobasal medium for trituration what will inactivate the papain? If I use the same medium for plating too, isnt glial growth affected without FBS? Perhaps I should use another medium for dissociation (like Jill's protocol), another for initial plating (with FBS) and another for cell growth.. Any thoughts?

Thanks a lot for all your comments till now!

Hi Vini,

I use BME media with FBS for the neuronal preparation and also seed neurons in the same media. Once the neurons get attached ( after 1-2 hour), I add the Neurobasal media (with B-27, PenStrep and glutamax).

This time, reason for cell death could also be because of papain (since you did not inactivate it).

Regarding the growth of glia, even if you do not add FBS, they still grow but at slower rate. and I guess you do not want high density of glia in your culture because after some time they start to kill neurons.

Good Luck

Sushma

Dear Vini,

It is critical that you inactivate the action of the papain.  Even if you think you have diluted it down for plating, it will continue to act on the cells. 

You mentioned in your original question that you see nice cells in the hemocytometer.   However, you don't mention whether you checked the viability with Trypan blue staining of the sample that you count in the hemocytometer.  I always used Trypan blue, and adjusted the plating dilution to the number of live cells that were Trypan Blue negative.  For me, that gave me much more reproducible results than just plating by the number of dissociated cells.  The number of live cells is dependent on the speed of your dissection and dissociation, as well as how gently you handle the cells.  We used to try to get the dissection and dissociation done in an hour.

And in answer to your question about different solutions at different steps in the protocol--as you have found from your own work, there are reasons to use a certain solution at a particular step.  Many people I know use the solutions in Gary Banker's protocol, which can be found in Nature Protocols (Kaech S & Banker G  2006  Nature Protocols 1(5) :2406-2415.) as well as in the book he edited with Kimberly Goslin.  With Neurobasal media and serum free supplements you don't need to go through the work of preparing astrocyte feeder layers for glial conditioned media to improve neuronal survival.  You can find out more about those protocols from Greg Brewer's work (Brewer, G.J. et al.  1993 J. Neurosci. Res. 35:567-576), or in the chapter he co-authored with Paul Price about neuronal cell culture: Chapter 19, by Paul J. Price and Gregory J. Brewer, in Protocols for Neural Cell Culture, 3rd Edition, 2001.  Ed.: S. Federoff and A. Richardson.  A link to an online copy is here:

http://www.bioon.com.cn/ewebeditor/uploadfile/201012/20101221133346840.pdf

In an answer to a previous question in a previous neuronal culture RG thread, Michael Wells from MIT has posted his protocols for postnatal cortical neuron culture.  They are very similar to what I have used for hippocampal neuron culture:

https://www.researchgate.net/post/Can_anybody_help_me_with_isolating_and_culturing_neurons_from_mice_brain

Good Luck!

Jill

Thanks, Sushma and thanks a lot, Jill for sending through the protocols. The protocol I am using seems extremely simplified and for my next round of cell culture I will try to include most of these things/protocols that you guys mentioned. Will post again here if things work (fingers crossed!).

Thank you...!

Vini, have you sorted this problem out? I'm currently having the same issue. I usually get 400,000 cells per mouse, and I see cells attached to the coverslip after 2h of plating, but in the second day after culture, cells are still rounded, and after 5 days, most of them are dead and there are just a few viable cells. So, I was wondering what problem you have had, 'cause it might be the same that I have right now.

Hi Fernanda,

I did sort this out with the help of the above suggestions. The problem you describe could be due to the coating. I am using 12 hours ornithin followed by laminin (laminin will help the processes spreading out). I also see this when my media is old, at least make sure you check the media pH. I also changed my trituration solution to include BSA and DNAseI and started getting more viable cells. Check out the protocols shared by Jill above. 

Good luck!