Planar Patch Clamp: Advances in Electrophysiology
13.3. Methods
For planar patch clamp the situation is different. After adding a cell suspension onto the patch clamp chip with a ∼1-µM hole, a cell is captured on top of the hole by applying suction. Since there is no relative movement between the sealed cell and the glass chip, no anti-vibration table, microscope or micro-manipulator is needed (Fig. 13.2b). Therefore, the Faraday cage can also be miniaturised.
Here we describe the functionality of the Port-a-Patch as an example for a planar patch clamp system. The Port-a-Patch (Fig. 13.2b) is a semi-automated patch clamp device providing data quality comparable to pipette-based patch clamping (4–6).
It utilises planar patch clamp chips (called NPC-1 chips), made from borosilicate glass, for the patch clamp recordings because of their excellent dielectric properties and clearly distinguishable stray capacitances. In the centre of the micro-machined glass chip is Fig. 13.2. Comparison of a typical pipette-based patch clamp rig on the top with a planar patch clamp rig on the bottom.
(a) demonstrates the complexity of a pipette-based patch clamp rig, including an anti-vibration table, a microscope, micro-manipulators, an amplifier, a computer and a Faraday cage. (b) The planar patch clamp rig consists of a Port-a-Patch, an amplifier and a computer. It has a much smaller foot print than the conventional rig.
an µm-sized aperture, onto which the cell is positioned auto-matically by application of suction (Fig. 13.1). Using a graphi-cal user interface, the suction protocol is pre-programmed and computer controlled. The software adapts the applied negative pressure according to parameters such as pipette resistance, series resistance, and slow capacitance. In this way, the programme can determine if a cell has been sealed to the chip and whether the parameters correspond to the cell-attached or the whole-cell recording configuration.
1. Typical cell lines, such as HEK 293 or Neuroblastoma cell lines, are grown in T75 flasks or plates of Ø = 96 mm and a surface of 60 cm2. The plating of the cells has an influence on the seal behaviour (see Note 2).
2. The cells are split every 2–3 days. The cells are kept under a confluence of 60–80%. It is important to avoid cell clusters (see Note 3).
1. Remove the medium from the cells and wash with PBS.
2. Add 2 ml of detachment buffer. Incubate for 2–5 min at 37°C.
3. Remove the cells with the medium from the plate and pipette them 3× gently up and down.
4. Centrifuge for 2 min at 100×g.
5. Discard the supernatant and re-suspend the cells in 1 ml of external buffer (resulting in a cell density of ∼1 × 106–5 × 107 per ml).
6. In our experience, the cells in suspension remain viable for up to 4 h, stored at room temperature.
1. Add 5 µl of internal K+ buffer to the inside of the borosilicate glass chip (Fig. 13.3a).
2. Screw the chip on top of the holder. The internal electrode makes an electrical contact with the internal solution.
3. Place the small Faraday cage in its designated space on top of the chip.
4. Add 5 µl of external buffer to the top of the chip. Now the external electrode (ground electrode) is electrically connected to the external buffer (Fig. 13.3b). The chip resistance and the offset can be measured.
5. When the PatchControl software is started, slight positive pressure is applied to the chip and the offset is corrected.
6. Before the application of 5 µl of the cell suspension, a slight suction of 50 mbar is applied. Once the suspension is added, a cell is attracted to the hole of the chip which leads to a small increase in the seal resistance.
13.3.1. Cell Culture
13.3.2. Cell Harvesting
13.3.3. Planar Patch Clamp Using the Port-a-Patch
7. The seal resistance is increased to a gigaseal by applying suction pulses and applying a negative voltage to the cells. This process runs automatically within the PatchControl software. It uses the same approach as an experienced patch clamper would do.
A rinsing of the cells with external solution also often improves the seal resistance.
8. The whole-cell access is achieved by short suction pulses.
For some cells it is helpful to support this process by zapping (see Note 4).
1. Giant Unilamellar Vesicles (GUVs) are prepared using the electroformation method (7) in an ITO-coated glass chamber connected to the Nanion VesiclePrepPro setup.
2. 20 µl of the lipid solution is deposited on the ITO-coated glass surface.
3. After total evaporation of the solvent the lipids are assembled in a dehydrated lamellate phase.
4. A greased O-ring is placed around the dried lipid film and 300 µl of a non-ionic intracellular solution, sorbitol, with a concentration equal to 210 mM, is carefully added to the lipid film.
5. The second ITO-slide is placed on the top of the ring, with the ITO-layer facing downwards.
6. The process of electroformation is controlled by the Vesicle PrepPro setup and all parameters (amplitude, frequency, duration, etc.) for the electroformation are programmed in the VesicleControl software. Typically, an alternating voltage of 3-V peak-to-peak is applied with a progressive increase for the rise time and a decrease for the fall time to avoid abrupt changes, which might otherwise rupture the formed GUVs.
The frequency of the alternating current is 5 Hz and is applied to the ITO-slides over a period of 2 h at room temperature.
13.3.4. Preparation of Liposomes
Fig. 13.3. (a) The bottom side of a patch clamp chip is filled with 5 µl internal K+ buffer with the help of a pipettor. (b) 5 µl of external buffer makes contact with the bath electrode on top of the chip. (c) The Port-a-Patch is covered with a small Faraday cage and it is connected to a USB controlled suction pump.
7. After successful swelling, the vesicles were stable over weeks to months, refrigerated or frozen.
1. Add 5 µl of KCl solution to the inside of the borosilicate glass chip (Fig. 13.3a).
2. Screw the chip on top of the holder. The internal electrode forms an electrical contact with the internal solution.
3. Place the small Faraday cage in its designated space over the chip.
4. Add 5 µl of KCl solution to the top of the chip. Now the external electrode (ground electrode) is electrically connected to the external solution (Fig. 13.3b). The chip resistance and the offset can be measured.
5. By starting the Patch Control software a positive pressure is applied to the chip and the offset is corrected.
6. Before the application of 5 µl of the GUV suspension, a slight suction of typically 10–40 mbar, is applied. A GUV is attracted to the hole of the chip and as soon as it touches the glass surface, it bursts and forms a planar lipid bilayer with a typical seal resistance of tens of GΩ.
7. The additional GUVs are removed from the chip by washing twice with 20 µl of KCl solution.
8. The holding potential is set to 100 mV.
9. 1 µl of the Gramicidin solution is added to the chip. Incorpo-ration of the Gramicidin channels is typically visible after 30 s to 2 min.
10. If no channel activity is visible after 3 min an additional µl of Gramicidin solution can be added.
The internal perfusion system consists of a special chip holder with an integrated internal perfusion and a perfusion system panel (Fig. 13.4). The internal perfusion system is an alternative head for the Port-a-Patch and can therefore be used with any Port-a-Patch. The perfusion system panel contains eight magnetic valves, which can be conveniently controlled by electrophysiological software.
1. For use of the internal perfusion with the Port-a-Patch, it is important that the internal electrode is connected to the ground (change the position of the switch on the back of the Port-a-Patch). This avoids noise and capacitance problems arising from the increased volumes of solutions for perfusion of the intracellular side (see Note 5).
2. The length of the fused silica capillary should be 1 mm above the inner plastic column as shown in Fig. 13.4 (see Note 6).
3. Proceed for obtaining the whole-cell configuration or the lipid bilayer as described under Subheadings 13.3.3 and 13.3.5.
13.3.5. Planar Lipid Bilayer Formation and Gramicidin
Incorporation
13.3.6. Internal Perfusion Using the Port-a-Patch
4. The internal buffer can now be exchanged.
5. Figure 13.5 shows an example trace of Kv1.3 currents endogenously expressed in Jurkat cells. The speed of the internal solution exchange depends very much on the access resistance (see Note 7).
Fig. 13.4. (a) For the use of internal perfusion with the Port-a-Patch a special measuring top has to be used. (b) For effec-tive noise reduction the internal perfusion is completely shielded.
Fig. 13.5. Example traces for an internal solution exchange in the whole-cell configuration. Kv1.3 currents are recorded from Jurkat cells with an internal solution containing high potassium. By switching to a caesium containing solution, the outward current is inhibited. Wash-out and wash-in could be repeated many times.
6. Figure 13.6 shows an example trace of Gramicidin currents in a lipid bilayer. In this case the internal perfusion is much faster.
7. It is important to mention that the final concentration of chloride ions should not be changed during an experiment without the use of an agar bridge or careful correction of the offset potential (see Note 8).
8. An exchange of the internal solution can also be done without the use of an internal perfusion system (see Note 9).
1. It is possible to use different detachment agents for harvesting the cells. In our experience, the standard trypsin/EDTA solu-tion works very well. Some people prefer Accutase or Accu-Max. Accutase is a mixture of a protease and a collagenase.
It is supposedly gentler on the cells, since no inhibition of the protease is necessary. AccuMax is a more concentrated Accutase, plus it has additional DNAse activity. The protein expression is supposed to be higher while using this reagent.
2. Some cell lines are more difficult to seal than others. It is possible to improve the seal rates by optimising the cell culture. We see better results in seal rates by using T75 flasks instead of much smaller Petri dishes. The time between splitting and the confluency of the cells are also important parameters to adjust. If cells tend to be very fragile, it is better to harvest