Growing adult neurons - (Aug/01/2007 )

Hi there,
I was wondering if anyone here has ever tried to grow adult neurons?
I have been playing around with the protocol published by Dr Brewer however my yield is always very low. The most I ever get is about 3 neurons per 35mm dish.
Any suggestions?

Thanks
-Weezie

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Well I don't know what your protocol is, but what are you growing them from and how?

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My final goal is to treat rats chronically with a drug, do some cognitive tests and the culture the neurons to look at axonal transport of mitochondria. We believe that this drug somehow interferes with axonal transport, possibly by altering motor proteins specifically those responsible for the transport of mitochondria.

The protocol requires removal of the hippocampus from the adult rat (of approx. 1-2yrs old), followed by slicing into 0.5mm slices then:
- papain
- trituration x3 collecting the supernatant of each trituration
- adding the supernatant to a density gradient called optiprep
- collect the neuronal fraction
- centrifuge to separate the debris from the neurons and microglia
- resuspend the fraction and centrifugation to remove density gradient solution
- plating neurons
- replacing media after one hour to remove debris

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Hmm....maybe time your trituration each 45 s each...don't know how you time this normally. Also make sure you use the right pipette (9 in. pasteur pipette, and flamed). If you already do these, maybe it is just too vigorous and you could try an experiment where you triiturate in one sample the same way you do, along side a second and a third where you become more gentle each time. Record exactly how you do it each time, ie how many seconds per trituration adn how many times per each 45 s interval. Then you can compare the results- it's really easy to break the cells at this point.

Good luck!

Also, out of curiuosity, KIF's or dynein? I work with a lot of KIFs and my lab does some similar research, which is why I ask

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Thanks I will def try that.
We are working with Kif. We think it is covalently modified by our toxin.
What is your interest with kif?

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