B27/Neurobasal medium for hippocampal neuron culture - (May/07/2007 )

Hi, anybody has run into trouble with Invitrogen's B27/Neurobasal medium recently? My cultures have always been good till two months ago. Invitrogen told me B27 quality is always inconsistant. Lots of people said buy several lot # and test them.
I didn't follow exactly Invitrogen protocol, I plated cells with BME+5% FBS first, then shift to Neurobasal in 8-12 hours. neurons survived well in BME, but all died in Neurobasal. Could this make it difficult for neurons if B27 quality is not as good as before?
Thanks!
Yicambridge

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I have been using neurobasal with B-27 and I change media after 2-3 hrs and so far they are fine. I am using it every week.

B27 is important for neuronal survival but we havnt seen much of a difference in last few months or so. I actually buy B-27 every 3-4 months. Once the invitrogen guys called us to inform that something could be problematic with B-27 but we never had trouble.

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I met the same problem with B27. Our lab is in Hong Kong, and actually there are more problems than reported in US (heard from one friend). We are trapped by B27 quality problem for almost a year and we actually switch to other systems to study.

For sure, the quality of B27 is of great importance. And you will find out later that some bench of B27 may result in quite obvious growth of glia cells, while some do not.

Good luck!

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there is always growth of glial cells in neuronal cultures. You have to use antimitotics to get rid of most of them.

We use them at 5 days and change media after 36 hrs.

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Hi,
I experienced many times that B27 didn't work. The result was always the same: neurons died
When we order a new lot we always check it.

Regards
Sylwia

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This may be a stupid question but how do you check that the B27 is good?

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Once invitrogen had told us that the batch of B27 we ordered might have some problems. But we never experienced any thing. Only if the cells (neurons) are not treated gently they will die. I donot think B-27 can be blamed. Primary neuronal culture is sensitive and every solution and every step while preparing them have to be assessed if one needs reproducible good cultures.

We had to change trypsin because we thought it was killing the neurons. Sometimes, coating the plates with the right stuff can make a difference. Once changing the poly-ornitine which we use to coat plates made a whole lot worse.

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I am sure that my poly-d-lysine is good, its from the same original batch that has made neurons grow in the past (nb. it is all stored at -20oC). The only thing that has changed is the B27.
GRRRRRRRRRRRRRRR!!!!

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