Fresh dissociated neurons --problems!

Ren-Shiang Chen crs at
Tue Oct 3 09:17:12 EST 1995

First time post here, any inappropriateness please forgive me.

I wonder if anyone can give me some suggestion about preparing fresh 
dissociated  rat hippocampal neurons.  My lab is proforming whole-cell 
recording on them for study drug kinetics.

Presently, I incubate the brain slices from 7-15-day neoborn Long-Evan
rats in a dissociation media (82mM Na2SO4, 30mM K2SO4, 2mM MgCl2, 2mM HEPES, 
pH7.4 by NaOH) containing 3mg/mL protease XXIII (Sigma) for 8-13 min. 
Then the enzyme treatment is stopped by the same media containing typsin 
inhibitor and albumin (1 mg/mL, both, Sigma). And then CA1 region is 
triturated to release single neurons before use.

Similar methods are seen in literatures.  Conditions of incubation such as
with/without 2mM CaCl2 and/or 2mM MgCl2, variable time of treat, 
concentration of protease: 2 or 3 mg/mL, had been found not optimal for my
preparation.  The cells do not seal tightly with my pipettes (I am sure 
that's not the fault with the pipettes).  Beside, the cells often do not 
attach to the bottom of the recording chamber, that make seal more 
difficult--often more than 200pA leak current.

A few questions:
1. Is protease XXIII (Sigma) stable under 0-4 degree(Celcius) storage ?
2. What's the longest time of incubation that will not damage the cells?
3. Any correction should be made to get better seal ?
4. Any strategy to enhace the attachment of the cells to the cell box?

Any suggestion appreciated, sincerely. 

   |\/\/\/|   Ren-Shiang Chen, crs at
   |      |    
   |      |      Dept. Physiology, College of Medicine,
   |  (O)(O)     National Taiwan University, Taiwan.
   C      _)     	
    | ,___|      		
    |   /    

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