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 37oC,
wash with HBSS for three times,
treat with papain solution (10unit/ml) 15min at 37oC,
Inactivated with 10% Horse Serum,
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.