Hi,

here is the protocol I used if that can help:

Cortices were isolated from 14 day-old mouse embryos in sterile Krebs solution containing 125 mM NaCl, 5 mM KCl, 1 mM NaH2PO4, 15 mM D-glucose, 25 mM HEPES, 0.05 mM BSA and 2 mM MgSO4. Cortices were then incubated for 15 min at 37°C in Krebs solution containing 0.1 mM trypsin (Sigma-Aldrich). Krebs solution containing 25 nM DNAse (Sigma-Aldrich) and 130 nM soy bean trypsin inhibitor (Sigma-Aldrich) was added to the suspension (1:1 dilution), and then centrifuged at 250 g for 3 min. The pellet was resuspended in new Krebs solution, and centrifuged again. The cells were finally resuspended in Neurobasal medium supplemented with 2% B-27 supplement and 0.5 mM L-glutamine. Neurons were plated on 48 well-plates at a density of 125 000 cells/well previously coated with 5 μg/ml poly-D-lysine and were used for experiments 6 days after plating. After 5 days, half of the medium was changed to complete Neurobasal medium.

Dear Angelica, my suggestion would be i) to check the lot of B27 you used before and try to get the same lot again (by asking around perhaps), ii) do you regulalry use FBS in you medium? If yes, do the same for FBS. Very often it turns out to be the new lot of B27 and/or FBS. If it turns out to be new, ask your supplier to provide you with the samples to test it in your culture conditions. Another small tip, we usually wash our coverslips with NBA instead of Water, but if you wash with the water, I suggest you check the pH of your dH2O. I hope this is helpful! Good luck!

Hi Angelica,

So, did you use to get healthy cultures, and this problem popped up suddenly? Obviously, you need to track if something in the pipeline was changed.

One of the most critical factors for neuronal attachment and growth in my experience is the type of glass coverslips; I highly recommend using the glass listed in the Kaech and Banker Nature Protocols paper (lists catalogue number for the glass for ordering from Fisher Scientific as 12-545-xx). I also recommend their prescribed way of treating the glass with nitric acid, before washing it and coating with PDL or the like. I personally did not allow the coverslips to dry after coating with PDL; I would either aspirate the PDL-containing buffer, wash, then replace with neuronal media, or if protocol requires empty wells, then I would leave the aspiration of PDL and washing of the wells until just before the neurons/media are placed in the wells.

Also, another suggestion (although I doubt this is the reason you're not seeing any neurite extension at all), is to eliminate the P/S (I'm guessing that's antibiotics?). Ideally, one would culture without antibiotics altogether, but if you're finding contamination a problem, perhaps leave out the antibiotics from your trituration solution, and only add antibiotics once you've plated the cells.

Good luck resolving this issue,

Hi Natalia, thanks for the help! I'll change the lot of FBS and see if that does anything but I previously was doing this protocol without the addition of FBS with the same issue. I used to wash with PBS and switched over to water but I just checked the pH and it's fine. 

Hi Haider, yes I used to be able to do these cultures with the Kaech and Banker protocol you cited but things went awry and I decided to modify the protocol a bit (to no avail). I have been plating my neurons in 6 well plates without coverslips (for the time being) but will try the nitric acid wash to see if that does anything. Thanks for the suggestions. Yes P/S is pen/strep I'll try eliminating and see how that works out. I just can't seem to pinpoint exactly where I went wrong. 

Dear Angelica,

If I understand the details of your protocol correctly, you just started adding FBS to your protocol. One of the functions of FBS would be to inhibit the action of the trypsin, which would manifest as increased cell survival.  Since trypsin is such a harsh enzyme (I personally prefer Worthington papain tested for neuronal tissue culture), I think you might get a better result if you substituted soybean trypsin inhibitor and BSA, as suggested by Sighild.

I also agree with Haider about the storage of the coated wells.  I personally think it is better to store the wells in Neurobasal without any supplements, or to aspirate the final wash just before plating.  Since you are washing with water rather than PBS, I suggest storing in media without supplements.  Any protein, such as those in FBS, that you add to the wells long term will bind out to the PDL.  If you leave that for too long, it is more difficult for the neurons to bind to the PDL during plating, as well as for the axons to grow out.

Good Luck!

Jill   

Thank you so much for your answers Jill and David. I fixed my issue! I changed the lot of FBS as well as B27 and glutamax (just as a precaution). I stopped using P/S in my media as well. Instead of diluting the PDL in dH20 I diluted in 0.5M borate buffer pH 8.5. I washed the plates with PBS instead of dH20 as well and as suggested I aspirated the very last wash right before plating. I also decided to do the trypsinization step in pure 0.05% trypsin without HBSS + hepes. I had originally being trypsinizing in 2.5% diluted down to .25% trypsin in HBSS + hepes. I think the trypsin change made the biggest difference. My neurons are now growing neurites again!

The only issue is that because I'm intending to take RNA from these cells I still see cells without neurites and these don't stain beta III tubulin positive. I've tried washing with PBS at different steps but I can't seem to get rid of these. I know it's difficult to culture purely neurons but are there any tips to get rid of these or to normalize for this? Thanks again for all the help! 

You could try FACS or MACS before RNA extraction.

Dear Angelica,

To further purify your cultures you may have to try some selection steps.   I have never tried this for cortical neurons (only retinal ganglion cells), but you may want to investigate the Miltenyi Biotec beads.  There may be other selection protocols that work for cortical neurons.  Links to a few of them can be found at this RG thread:

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

Good Luck!

Jill

Hi again Angelica,

My understanding is that the Banker protocol, where the triturated cells are seeded onto coverslips, and shortly thereafter they are transferred to a new well with conditioned medium was devised to do just that - separate neurons from other cell types, especially glial cells. The idea is that the neurons attach to the coverslip more quickly than glial cells (but don't know about fibroblasts), so if you transfer the coverslip in less than two hours to a new well with pre-seeded glial feeder layer (and suspend the coverslip face-down using paraffin dots), you should wind up with a more pure neuronal culture. Should also include mitotic inhibitors to curb proliferation of other cell types.

Its a bit late to the party, but in case you were wondering why it was going wrong. In the US, water comes from copper pipes. Even the nanopure water etc can contain free metal ions which result in neuron stress and death. If you use nanopure water instead of molecular grade water, your neurons will not grow. You won't detect a pH difference because that isn't the issue.

Sometimes you are doing nothing wrong and instead it is your geographical location. It could be something happened to the pipes in the building. I noticed you are from Yale and were washing with water. Sometimes you have to be godlike to figure out all the things that can go wrong independent of the experimenter. The free ions in water is documented for obtaining functionally intact mitochondria in other fields. Moreover, we had a similar problem. It sounds silly but it is true.

Just to add to Pj Flannery's comment about water sources-we didn't have any problems with the building deionized water source (which feeds our Nanopure system), but we also had the Rep test the water source and recommend the cartridges for our system.

Instead, the water used by the autoclave may be a problem for many people who process their own glass cover slips for neuronal culture. We invested in a small autoclave that used distilled water rather than using the large molecular biology autoclaves in the shared facility.

Yes, it can be frustrating to keep all the variables under control, especially when companies change or discontinue reagents!

Cheers!

Jill