Biology Reference
In-Depth Information
the reference barrel (under pressure) to prevent silanization of the reference barrel
(which would result in both barrels being Ca
2
þ
-sensitive).
The larger tips of these electrodes make the filling easy. Both barrels can be
easily backfilled. The silanized barrel is backfilled with a reference solution con-
taining 10 mMCaCl
2
and 100 mMKCl and the nonsilanized barrel with a solution
containing 140 mM NaCl. A column of the neutral Ca
2
þ
ion-exchange cocktail
ETH 1001 or 129 (Fluka Chemical, Ronkonkoma, NY) 50-250
m
m long is easily
drawn into the silanized barrel. Ca
2
þ
electrodes with these tip diameters exhibit
Nernstian behavior over the range 10
m
M-10 mM Ca
2
þ
(
Bers and Ellis, 1982
).
The resistance of these MEs was typically 1-5 G
O
for the Ca
2
þ
-sensitive barrel and
1-4 M
O
for the reference barrel.
The impedance of the two barrels is very di
erent, but their fast response allows
relatively rapid interstitial [Ca
2
þ
] monitoring. To match signal response kinetics to
voltage steps a variable-passive R-C filter can be added to the reference barrel
signal after the signal has come from operational and o
V
set amplifiers. This filter is
adjusted while a square voltage pulse is fed into the bath until the best matching
with the Ca
2
þ
barrel response is obtained (
Bers, 1983
). These Ca
2
þ
electrodes
typically exhibit Nernstian behavior at least over the range 10
m
M-10 mM Ca
2
þ
.
This is satisfactory for typical extracellular [Ca
2
þ
] measurements and the double-
barreled electrodes are easier to calibrate and use than intracellular impalements
(described in more detail below).
V
6. Calibrating Bath and Solution Perfusion
It is preferable to calibrate ion-selective MEs in the experimental chamber in
which measurements are made or to have the calibrating bath as close as possible in
design and proximity. Our calibration chamber is a ''flow-through'' type (volume:
0.1 ml), immediately adjacent to the experimental chamber. Note that it is conve-
nient that the experimental chamber is viewed from the front, and not from above.
7. Calibration Procedure
The bath electrode is either an Ag wire (chlorided by dipping it in bleach for
15-20 min) or an agar bridge. Ideally, a conventional electrode (3 M KCl filled)
should also be immersed in the bath, and the di
erential voltage (ion-selective ME
minus conventional) should be read. We use a commercial amplifier (FD-223 from
WPI) or a home built amplifier using varactor bridge preamplifiers (AD311J,
Analog Devices) as described by
Thomas (1982)
.
We have adopted the following method to quickly select suitable MEs. The ME
is mounted in its holder and advanced into the calibrating bath, allowing the trace
to stabilize. If the device used to measure the signal has a resistance measurement
feature, it is worth measuring this parameter. In fact, our experience has been that
for the sharp Ca
2
þ
MEs having resistances ranging between 100 and 250 G
O
were
suitable for our experiments, in terms of linearity and detection limit of the
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