Biology Reference
In-Depth Information
3. Electrolyte Filling of the ME
The filling or conducting electrolyte is introduced in the ME from the back
(back-filling), but because of the hydrophobicity of the glass wall (due to the
silanization), the tip does not get filled immediately. The strategy to fill the ME
completely depends on the absence or presence of the ligand at its very tip.
In the case where the ME is first filled with the electrolyte, the back of the ME is
connected, via a flexible tubing, to a 50-ml syringe, whose plunger has been
previously pulled to 5 ml. Positive pressure is then applied and, as assessed by
microscopic observation, the electrolyte creeps along the ME wall and fills the tip.
The biggest air bubbles are then removed by gently flicking the ME, held tip down.
Although we have initially applied this procedure to filamented MEs, a similar
success rate (more than 90%) has been obtained with nonfilamented MEs
(surprising, because silanization is expected to limit aqueous filling ease).
Whatever the method used, if the electrolyte column was interrupted by air
bubbles then, gentle heating of the tip of the ME, under microscopic control, with a
tungsten-platinum wire, according to the device described by
Thomas (1982)
, can
remedy this problem. Once filled with electrolyte to the tip, the hydrophobic ligand
can be introduced (see below).
In the case where the electrolyte is added after the Ca
2
þ
ligand, additional
problems exist. In fact, caution has to be taken to avoid the presence of air at the
electrolyte-ligand interface. If a traditional whisker is used, great care has to be
taken not to accidentally disrupt the column of ligand, which can lead to mixing of
oil and water, making unstable ion-selective MEs. If a heating filament is used, care
has to be taken not to heat the ligand because it is likely that the local high
temperature may damage the ligand properties.
We have used a filling solution that has an ionic composition mimicking the
intracellular medium (in mM): Na
þ
: 10; K
þ
: 140; HEPES: 10; EGTA: 1; pH 7.1
(at 30
C) and pCa 7. This solution is in fact identical to the calibrating solution
having the same pCa (see below and also
Orchard et al. (1991)
) for additional
comments).
Our experience has been that it is best to minimize the time between electrolyte
and ligand filling. We prefer to fill the ME with the ligand as soon as the ion-
selective ME is filled with the electrolyte (although we managed to draw the ligand
in the tip 2-3 h after electrolyte filling). If MEs are left overnight with the filling
solution, it can be very di
cult to draw ligand into the tip; this observation could
be explained by a progressive glass hydration, causing it to lose its capability to
retain the ligand.
Y
4. Preparation and Use of the Ca
2
þ
-Selective Ligand
For repetitive and long-lasting Ca
2
þ
measurements with Ca
2
þ
-selective MEs
dissolving the Ca
2
þ
ligand in a ''cocktail'' containing PVC is useful (
Tsien and
Rink, 1981
). Although the cocktail available from Fluka (Cocktail II containing
