Ilooked over your protocol and several things jumped out at me. First,
2.5 mg/ml of trypsin is WAY too much. Was this a typo? You should be
using 0.25 mg/ml or less. Batches of trypsin can vary considerably--
sometimes we have to dilute the trypsin stock two-fold because we kill
cells when we dissociate. Also, make sure the solution you use for
trituration contains serum or soybean trypsin inhibitor. Trypsin is
very sticky to cells and need to be inactivated before you triturate.
Beware of DMEM!! It contains ALOT of glutamate-- not good for cortical
neurons. Try MEM or Neuro-basal medium (Gibco-BRL).
Our experience is that cortical neurons do much better in serum-free
medium-- many batches of serum are toxic for CNS. Fetal calf serum in
particular varies wildly from lot- to lot. If you must use serum,
pre-test different lots for optimal cell growth and viability. Instead
of serum we use B-12 supplement (Gibco BRL) or N-2 (original
Bottenstein and Sato serum free supplement).
Rae Nishi, PhD
Dept. Cell & Developmental Biology
Oregon Health Sciences University
nishir at ohsu.edu
**that's Orygun, NOT Ora-Gone**