Establishing a Whole-Cell Configuration through the Patch Clamp Method

Using the fine focus wheel of the microscope, start focusing down while lowering the micropipette gradually. Always focus down first, and then, lower the micropipette to the plane of focus to ensure that the micropipette tip does not abruptly penetrate into the slice. When the micropipette comes in contact with the surface of the slice, slow down the micromanipulator speed to medium-low mode. Gently apply light positive pressure to clear any debris on the path.

Then, approach the cell, either by alternating with the X, Y, Z control knobs, or by approaching diagonally where both XZ axes are changed with the rotation of the Z-axis's knob. When the micropipette is close enough to the cell, a dimple will appear on the cell surface.

Now, apply a weak and brief suction through the tube that is connected to the pipette holder suction tube in order to create a seal. While a gigaseal is forming, use the computer-controlled amplifier commander to bring the cells' holding potential to the physiological resting potential in order to prevent sudden changes once the membrane is ruptured. After the gigaseal has formed, compensate for the fast or slow capacitance.

Here is very important that the suction applied is not too strong, otherwise the membrane may rupture before establishing seal.

If the seal remains stable, and above 1 giga ohm, apply a brief and strong suction to rupture the plasma membrane.

The suction must be brief, and is stronger than the pressure applied when establishing a seal in order to properly rupture the membrane and achieve a stable whole-cell configuration.