hippocampal cell culture - I need a help (Sep/13/2006 )
Hi all there. I am new in cell culture and dont know anything about cell shape. My cells are spherical after isolation and their shape dont change even after 3-4 days. I like to know the normal pattern of hippocampal cell growth in cell culture. Meaning that how much time last to cells getting their projections.
I dont exactly remember the time course of the hippocampal neurons but they should have flattened out by 3-4 days. U should c some decent processes outgrowth by 1 week.
We would usually do experiments with these primary cultures after 1 week.
Hi Saya. I am interesting in do some hippocampal neurons culture, can you give me some tips about it???? or some protocols??? that i can read.
How old are the fetus? If it is older than 18d, it can not be sphere.
its difficult to dissect out the hippocampi if its older than E18. But one could still get some hippocampi.
Hi MCR. This is my protocol:
Bring 18-19 day old embryos ( kill the pregnant rat using CO2 and remove the embryos using sterile instruments).
Transfer the embryos to a big dish containing CMF HBSS solution.
Cut the heads and transfer them to a petridish containing that solution.
Using small scissor cut open the skulls going through the foramen magna and cut all the way and at the end cut sideways to open the skull (a Y shape cutting) to expose the brain.
Then try to remove the brain by pushing it gently thorough lambda to bregma.
Transfer all brains to another petridish containing CMF-HBSS.
U must seperate the 2 hemispheres and try to expose the hippocampus and dissect around it and then transfer it to CMF-HBSS media.
You can accumulate 10 hippocampi in a conical tube and add trypsin 2.5% to your solution in a way that your final trypsin concentration must be 0.25%.
You must put the conical tube in the incubator with 37 c for 10-15 min.
Stop trypsin using a few drops of FBS and triturate using fire polished pasteur pipette.
Let the cell suspension to settle big clamps for 2-5 min. Remove the supernatent using a pasteur pippet and cetrifuge it. resuspend the pellete in 2-4 ml of the media and count the cells using trypan blue to see dead and alive cells.
Seed the cells 100000 cell per each cm2.
Feed the cells 2-3 in a week.