Serum free hippocampal slice cultures - (Feb/19/2007 )
Hello All!
I would like to know if anyone out here has experience in serum free slice hippocampal slice cultures? I have been growing slices well in serum containing medium but now need to do some experiments without additional serum. The way I do it is to grow them in serum containing MEM for 3 days and then switch to Neurobasal supplemented with 2% B27, glutamine, 25mM glucose. But the slices invariably die. Could someone help me?
-K
I used do hippocampal explant cultures often. How thick are your slices? and are you giving them enough time to clear. that is to say in the first 4-6 days there will be a great deal of cell death (visualized as lots of dark spots) but by about a week, the slices should look relatively free of these dark spots. it is at this time that we begin weaning our explants off of serum. so up until this point the tissue has been in MEM with 10% HS and L-glutamine, dextrose, & pen-strep. Then on day 1 of weaning, we switch the media to Neurobasal with 5% HS, B-27, dex, L-glu, & p/s (we call this Step 1 media). ~24 hours later, we switch to step 2 media (Neurobasal with 2.5% HS, B-27, dex, L-glu, & p/s). 24 hours after that we switch to step 3 media (neurobasal, b-27, dex, l-glu, & p/s). We then leave them in step 3 for at least 48 hours before beginning any experimental treatments. They will remain in step 3 media from here on out. I am not at the lab right now, so I don't have access to my protocols, but I think the information I gave is accurate. Also we grow our explants on membranes from millipore.
Let me know if this helps or if you need more information/help or if want the typed up protocol. I'll be glad to oblige.
I would like to know if anyone out here has experience in serum free slice hippocampal slice cultures? I have been growing slices well in serum containing medium but now need to do some experiments without additional serum. The way I do it is to grow them in serum containing MEM for 3 days and then switch to Neurobasal supplemented with 2% B27, glutamine, 25mM glucose. But the slices invariably die. Could someone help me?
-K
Just curious, why do you use HS, instead of FBS?
I asked my PI the same question when I started working with explants, he told me that a post doc in the lab before my time had optimized the procedure, and that he found HS to work best.
Thank you Mondo..I have been grading the serum from Day 4 on and have slices slightly better than before.I will try weaning from week 1 on and let you know how it goes.
Thanks again