Neuronal Cell Culture - Media Changes - How to change media half and half (Jan/05/2011 )

So I have recently started a postdoc in which I'm working on setting up cortical neuronal cell culture for the first time. I had some pretty basic questions I would hope someone could answer. It is my understanding that after plating the neurons only half the media should be changed every 3-4 days. However, some of the media inevitably evaporates over the 3-4 days the neurons are incubating. So how to you handle the media changes? Seems to me that there are two ways to do this, both of which also appear to have potentially critical issues:

1) Do you get an approximation of how much media is left using a pipetter and then only remove half of that and replace it with fresh media? The problem I see with this is that eventually you would have very little media left after 3 or 4 replacements. Do you then put a cap on the minimum amount of media?

2) Do you "top up" the media to the appropriate volume (e.g, 2 mL in a 35mm culture dish) while making sure to never to add more than half of new media? The problem I see with this is that there would then be an increasing concentration of what is in and added to your media while the H2O evaporates out.

Any advice on this would be much appreciated!

Thanks.

MM

Just top it up.

But if you use neurobasal media+ B27, then you donot need to change media every few days for cortical neurons.

scolix on Sat Jan 8 19:57:48 2011 said:

Thanks for the response...I do use neurobasal + B27 + glutamate. Why do I not need to change media every few days? Sorry I'm a bit new to this whole in vitro business.

Thanks

MM

Regarding evaporation of your media: practically all types of plastic have some empty spaces which you can feel with sterile water to prevent evaporation. You can even fill with water Labtech chamber slides. I also place them in 150-mm Petri dishes and pour some water into the dish. I almost don't have any evaporation. Good luck!

katenkak on Thu Mar 17 12:57:59 2011 said:

Thanks for the input, however, I plate in 35mm dishes that don't really have any place for H2O. I would use 6 well plates, but I'm interested in manipulating my dishes individually and want to minimize how often I remove my cells from the incubator given their temperature sensitivity...