USE OF THE PATCH CLAMP – A TUTORIAL
•
35
Chapter 3
Whole-Cell Recording (Real Cell)
Set the front panel controls of the Axopatch 200B as follows:
PIPETTE OFFSET: About 5.0
ZAP: 0.5 ms
SERIES RESISTANCE COMP. % PREDICTION: 0 %, OFF
SERIES RESISTANCE COMP. % CORRECTION: 0 %, OFF
SERIES RESISTANCE COMP. LAG: 1 µs
WHOLE CELL CAP.: 0 pF, OFF
SERIES RESISTANCE: 0 MΩ
HOLDING COMMAND: 0 mV, toggle x1, OFF
SEAL TEST: ON
METER: Set switch to I
MODE: TRACK
CONFIG.: WHOLE CELL (β = 1)
OUTPUT GAIN: α = 10 or as desired
LOWPASS BESSEL FILTER: 1,2, or 5 kHz
LEAK SUBTRACTION: ∞ MΩ, OFF
Insert the pipette and its holder into the input connector of the patch clamp. In this case,
the pipette should usually contain a filling solution that is low in Ca
2+
and the tip should be
as large as possible to minimize the pipette resistance (the importance of low pipette
resistance is obvious from the previous series resistance compensation discussion). At this
point, proceed to insert the holder, lower the pipette into the bath and make a seal in the
same manner as described in the preceding single-channel recording tutorial.
A pulse of strong suction is required to rupture the cell membrane. This can again be done
by mouth suction or by a syringe. If you are using a 10 ml syringe, disconnect the suction
line from the syringe end briefly and push the plunger most or all of the way in. Reconnect
the suction line. Draw back slightly on the plunger until a large capacitance transient
suddenly appears. An example of this is shown in Figure 9. Again, many schemes of
rupturing the cell have been used by investigators.
To Next Page
Loading...
Need help?
General
