rat primary hippocampal culture - (Mar/01/2012 )

Hi all,

Recently, I am having trouble to get heathy rat hippocampal neurons. The protocol has been set up for 4-5 years. I always follow the same protocol, and I could successfully get heathy neurons and culture they more than 20 days. However, during past two months, neurons dead at DIV1. And they hardly survived more the 4 days. Attached picture was took at DIV3. I try to use all newly prepared solution, but nothing helped. Hope somebody can help me.

here is my protocol:
Dissect E18 rat hippocampus in NB buffer at 37^oC,
wash with HBSS for three times,
treat with papain solution (10unit/ml) 15min at 37^oC,
Inactivated with 10% Horse Serum,
remove papain,
Trituration in MEM (5%HS, 5%FBS) with 10ml pipette for 50 times
plate ~2000cells/mm² on PLL pre-coated coverslips
incubate at 37C, 5%CO₂
after 24hr, change half medium with NB+ B27+ 0.4 mM Glutamine
add 5 uM cytosine- -D-arabinofuranoside at DIV3
at DIV4 change half medium with NB+ B27

Any help or suggestions would be highly appreciate.
Thanks in advance.

Hi! I'm from STEMCELL Technologies and we have heard of a number of people having this problem. It could be due to variability in B27 supplement. See this news article in Science:
http://www.nature.com/news/2009/090506/full/459019a.html
You could try our NeuroCult SM1 supplement as an alternative - its is based on the original B27 formulation, but optimized for consistency and reliability. Hope it helps!
http://www.stemcell.com/en/Products/All-Products/NeuroCult-SM1-Neuronal-Supplement.aspx?utm_source=external&utm_medium=forum&utm_campaign=bioforum