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Guide to Electrophysiological Recording
common mistake is to assume that this Rs error is small, so as to avoid the fiddly
process of setting Rs compensation. This is false economy. Rs errors can be
surprisingly large and can easily render your hard-won data meaningless. We
strongly recommend that Rs compensation be used, at least to convince yourself
that its use is unnecessary in your particular case. The theory and practice of Rs
compensation are described in Chapter 5,
SERIES RESISTANCE
COMPENSATION
.
The Leak Subtraction feature of the MultiClamp 700B allows you to subtract linear
leak currents from the membrane current traces. Generally speaking it is not a good
idea to do this in the whole-cell configuration, because whole cells may contain
background currents that have some dependence on voltage. Software packages
like pCLAMP allow a user-specified after-the-fact leakage correction, which is a
much safer procedure.
Perforated-patch Recording
With some cells it has proven nearly impossible to go whole cell without loss of
seal. If you have one of those cells, you might consider the “perforated patch”
technique. In this approach, the very tip of the pipette is filled with a normal filling
solution and the rest of the pipette is backfilled with the same filling solution to
which 120-150
µ
g/ml of the pore-formers Nystatin, Amphotericin B or Gramicidin
[from a stock solution of 30 mg/ml in DMSO] has been added (Rae
et al
., 1991;
Yawo & Chuhma, 1993). Gramicidin has lower conductance than the other two,
but it offers the advantage that it is impermeable to chloride ions, which may be
important in some applications (Ebihara
et al
., 1995). A cell-attached seal is then
formed on the cell. Over a 5-30 minute time period, myriad tiny cation-selective,
voltage-independent channels are inserted in the membrane patch. These channels
allow small ions to equilibrate between the cell and the pipette allowing the cell to
be voltage clamped through the open channels. Since substances as large as, or
larger than, glucose will not permeate these channels, cell contents are not washed
out as in standard whole-cell techniques. This is an advantage or a disadvantage,
depending on the experiment. A distinct advantage is the maintenance of the
intracellular environment that might influence conductances. With the perforated
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
