[Neuroscience] whole cell recording from dispersed neurons

Bradley M. Cooke b-cooke at northwestern.edu
Fri Mar 31 14:43:52 EST 2006


Greetings,

Does anyone on the list have experience recording 
from 1º neuronal cultures? I am having a great 
deal of difficulty keeping my cells alive after 
their transfer into Tyrode's solution. Within 
about 20 minutes, they are all dead.

The composition of the Tyrode's (in mM) is as follows:

NaCl 150, KCl 4, CaCl2 2, MgCl 2, HEPES 10, 
Glucose 10, TTX, 0.50. Osmolarity: ~ 310, pH 7.3.

I have tried 4 different things in an effort to 
keep them alive: 1) bubbled the Tyrodes with O2 
prior to recording; 2) adjusted the osmolarity 
downward to approximate that of their growth 
media, which is Neurobasal + B27 and whose 
osmolarity is ~ 200; 3) Adjusted the pH to equal 
that of the Neurobasal, which is 7.0; and 
finally, made a mixture of 1:1 Neurobasal/B27 and 
Tyrodes and left the cells in that mixture for 
several minutes before transferring to pure 
Tyrodes.

All of these ideas have failed to prevent the rapid decline of the cells.

Any suggestions to improve my success rate would be greatly appreciated.

Many thanks in advance,

Bradley M. Cooke, Ph.D.
--

Department of Neurobiology & Physiology
2205 Tech Drive, Hogan 2-160
Northwestern University
Evanston, IL 60208
http://homepage.mac.com/bradleymcooke/index.html

p: 847/491 3027
f: 847/491 5211


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