Hello primary neuron experts!
We culture primary rat cortical neurons (E18) since decades in our lab. Never had we experienced a problem like now. The neurons seed well, evenly distributed single cells. After 2 or 3 days in culture they start migrating towards each others and within a week they form the crazy balls like you can see in the attachment. We did start a new lot of Neurobasal and B27. We did contact the company, but they were not able to confirm any problems with that batch. The problem occurs on both: PDL coated 6wells, and glass coverslips. What could it be? Suggestions welcomed! Help needed! Thanks in advance!
Hello--- check out this thread: https://www.researchgate.net/post/Why_my_Primary_Neuron_Culture_often_Weave_together
Thank you. But they do not really have a good answer there either. We can rule coating out. Since the very same coated 6-wells worked before without a problem.
Although you say it could not be so, your coating appears to be the problem, not the neurobasal or the B27. Try coating with high molecular weight poly-D-lysine (70K-150K or 300K) dissolved overnight in either PBS or Borate buffer pH 8.4. If you see lumps, the PDL is still not totally dissolved. It has worked miracles for us...
I have had similar problems, although not usually this severe. I am pretty sure it has to do with the coating of the wells or coverslips. I am using poly-D-lysine coated Permanox plastic chamber slides to culture cortical neurons from 15-17 day mouse fetal brains. The PDL is at 0.1 mg/ml in borate buffer pH 8.4. We don't see 'lumps' in the coating. These neurons do not seem to grow well on glass slides or calf skin collagen coating. In my hands, the neurons on PDL usually stay spread out and healthy for 11-12 days. But neurons in a recent set of cultures started to aggregate after 5-6 days. I suspect the PDL coated wells as they were in the 37C incubator for 6 weeks before use. I am going to try storage at 4C after coating, as others recommend it.
I now only use freshly PDL coated (less than 5 days) plates and they work best. In the past, I have had problems with plates stored for 2 week at 4C and used in parallel with freshly coated plates. Also coating at a basic pH ([email protected] or Borate buffer [email protected]) improves the coating. But even then, although rarely, there are problems with even a fresh coating. Is the PBS or Borate buffer or the PDL solution contaminated? Not quite as easy as making coffee. LOL
Elizabeth, That is interesting. I don't think the PDL or borate solution is contaminated. We don't get any bacteria or mold growing in these cultures.
I use Permanox chamber slides. The neurons like these better than glass. I have two slides coated in July and two slides coated this week. Those coated this week are at 4C and those from July are at 37C. However, we don't have a pregnant mouse this week and may not next week. Have you ever tried re-coating a culture vessel that was originally coated more than 5 days earlier? The slides are very expensive and I don't want to throw them away. Thanks!
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