HEKA Elektronik EPC 10 USB, Руководство по оборудованию, Страница: 61 / 81

10. Using the Patch Clamp
We give here a brief description of the techniques for establishing a seal and recording from a membrane patch or
from an entire small cell.
10.1 Forming a Seal
10.1.1 Initial Setup
The object is to apply voltage pulses to the pipette and observe the current monitor signal on an oscilloscope to
monitor the pipette resistance. A convenient way to set the desired adjustments of the amplifier is by activating
the
SETUP mode. The approach to the cell membrane and the formation of a giga-seal cause the resistance to
increase, thereby reducing the currents. A convenient pulse amplitude is 5 mV, which will cause 1 nA to flow in
a 5 MΩ pipette. For observation of the current pulses, it is convenient to pick a
Gain setting and Oscilloscope
sensitivity such that the current through the open pipette is reasonably sized. Other recommended settings are
also preset in the SETUP mode: The measure mode is set to
Whole Cell (voltage clamp) and V-Membrane = 0;
Filter 1 is set to 10 kHz and Filter 2 to 2.9 kHz, Rs Comp to ”Off”, C-slow Range to ”Off”. Before the pipette
is inserted into the bath, the current trace should be flat, except for very small capacitive pulses due to the stray
capacitance of the pipette and holder.
10.1.2 Entering the Bath
The surface of the solution is relatively ”dirty”, even if (as we strongly recommend) you aspirate some solution
from the surface to suck off dust and contaminants. For this reason it is important to apply a small amount of
positive pressure to the pipette before you move its tip into the bath, and also to avoid going through the air-water
interface more than once before forming a seal. When you do move the pipette tip into the bath, the current trace
may go off-scale (check clipping); in that case, click on the
Auto V
0
button or reduce the Gain until the trace
reappears. From the size of the current response to the Test Pulses , the pipette resistance can be calculated (the
pipette resistance is displayed in the EPC 10 USB window as R-memb ). If there should be no change in the trace
upon entering the bath, check for an open circuit, for example: 1. a bubble in the pipette; 2. faulty connection to
the probe input; 3. bath electrode not connected.
There is invariably a small offset potential between the pipette and bath electrodes. The V
0
control is designed to
provide a bucking potential to cancel this offset. With the pipette in the bath, select the Auto- V
0
button.
10.1.3 Forming a Gigaseal
When the pipette is pushed against a cell, the current pulses will become slightly smaller to reflect the increasing seal
resistance; when the positive pressure is released, the resistance usually increases further. Some cell types require
more ”push” from the pipette than others, but an increase in resistance of 1.5 (i.e., a reduction in the current
pulses by this factor) is typical. Application of gentle suction should increase the resistance further, and result
(sometimes gradually, over maybe 30 s; sometimes suddenly) in the formation of a gigaseal, which is characterized
by the current trace becoming essentially flat again (hyperpolarizing the pipette to -40 to -60 mV often helps to
speed the seal formation). To verify gigaseal formation, increase the
Gain to perhaps 50 mV/pA; the trace should
still appear essentially flat except for capacitive spikes at the start and end of the voltage pulse.
10.2 Cell-Attached Recording
You can now use the SEAL button, which will automatically adjust C-fast and τ -fast controls to minimize the size
of these spikes (some improvement can sometimes be obtained by manual adjustment). Transient cancellation will