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
