I was wondering if anyone has experience maintaining adult mice cerebellar slices in culture for more than 1 week. I have been trying to start some culture of slices but after 3-4 days, the slices starts to break up. I am using Neurobasal Media with B27, 0.5mM L glutamine and bFGF supplements. I am not sure if it is the age of the mice used that the cultures are having difficulty taking off? I am at the moment waiting to try some young mice, however, we cannot use mice less than 2-3 weeks old.
Hi, I couldn't but notice that you did not mention in your medium any antibiotics, especially for long term culture by experience I saw that they do improve the result and the reason why yyour slices break could actually be because of something else inside the slices. (Did you notice also if your medium becames a bit more yellow after a while? Acidification of the medium migth be a sign of fermentation from bacteria). I use cyprofloxacin (1:5000 from commercial stock) and pen/strep (1:10 from commercial stock) in the neurobasal medium added with L-glut, B-27, N-2 (1:10 from commercial stock)
Hi Elia
Yes I do have the standard antibiotics(PEN/STREP) and antimycotic (FUNGIZONE) in my culture media...
Mhh...what else then...the vibratome that you use in your lab is particularly dedicated to fresh tissue cutting or do you also use fixed tissues? In the second case, did you wash carefully the vibratome before using it for fresh tissues?
Hi Elia
The vibratome we use is only for fresh tissue culture only. Our lab is mainly an electrophysiology lab, hence strictly NO FIXATIVES allowed. All labwares, pipettes and instruments are strictly fresh culture use only. If I have youngish mice (2weeks old) I will try culturing them this week. ... thinking of both slice and also dissociated culture... I am not sure, but for your cultures, do you use very young animals. I had started with 8wks old mice... as that is the most ideal condition for my experiments... however, if I cannot maintain them in culture for more than a week, am now reverting to young mice. Thanks..
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