Hippocampal slices - How to know if they are helthy (Sep/01/2008 )

Hi people!

I have a question regarding organotypic cultures.
Usually, I kill the animal (7-10 days old), extract the brain, dissect the hippocampus and chopper it to 400um. Then I culture it on inserts (I use normally milliceli pic 3050) with a MEM/HBSS + serum medium. I assume that if I work quickly and clean the slices will be fine. But after a question of a college I started to wonder how do I know how healthy my slices are after they are taken. Even when I have treated and controls I don't know if they both are equivalent before start.
Goolging a little bit I found Propidium Iodide (PI) as a technic feasible to be used to "see" dead cells in acute. I tried and, to my eye, was not very specific...

Does anyone uses PI regularly?
Anyone can suggest any other method to check the slices "happiness"?

Thanks in advance

Seba

Heya,

I ususally culture hippocampal slices from P1 mice in a similar way than you, but they are 250 um thick. Around the third DIV, some areas (specially the center of the slice and the border between entorhinal cortex and hippocampus) become duller, and you can see a kind of opaque aggregates on the surface - that's normal, I think they're just glia covering the surface of the culture. They will eventually disappear, and on 8-10 DIV your slices should appear more translucent, with a brilliant surface.
I don't use Propidium iodide to check the health of the cultures, but later on, when I perform immunodetections on the cultures, I can see if they were helthy or not - if dissection takes too long (that's a crucial factor for their health) then you can see dead cells in the immuno. 1hour and a half or 2h since killing the animal to planting the slice is the maximum I would accept to have healthy cultures.
I want to remark that your cultures are thicker than mine, and that's an important issue you have to bear in mind: the thicker the slice, the more hipoxia (even anoxia in the core of the slice) they will suffer from, and that means death.
Concerning the culture medium: do you add supplements such as B27 and N2? That would help, too.
I hope this helped you, and good luck.

Cheers,

Hi SaRa,

fist of all, thanks for the answer, nobody around the lab work un hipp culture and I dont get too much feedback...
I'm using a MEM+ HBSS+ serum media... but I would like to try with B27 of N2. At wihitch concentrations should I use it, 2% would be ok?
On the other hand, if I use this supplements, should I take out the serum?

Thanks again

Seba

Hi SaRa,

fist of all, thanks for the answer, nobody around the lab work un hipp culture and I dont get too much feedback...
I'm using a MEM+ HBSS+ serum media... but I would like to try with B27 of N2. At wihitch concentrations should I use it, 2% would be ok?
On the other hand, if I use this supplements, should I take out the serum?

Thanks again

Seba