Jonathan D. Lippiat
11.3. Methods
11.3.1. Cell Culture
11.3.2. Preparation of Amphotericin B Solutions
11.3.2.1. Stock Solution
1. Measure 2 ml of the pipette solution into a clean small glass beaker or vial of approximately 10 ml capacity (see Note 5).
2. Pipette and dissolve 8 µl of the 60 mg/ml stock amphotericin B in DMSO into the pipette solution to give a final concen-tration of 0.24 mg/ml (see Note 6).
3. Wrap the beaker with two layers of aluminium foil and place on ice until use (see Note 7). This solution is viable for 3–5 h.
Remove a dish of cells from the incubator, replace the culture medium with 1–2 ml of the bath solution and place on the micro-scope stage.
A schematic of the following steps is shown in Fig. 11.1.
1. Pull patch pipettes from the glass capillaries using the puller and polish using the microforge. The resistance in the experi-mental solutions should be 3–5 MΩ (see Note 8).
11.3.2.2. Amphotericin Pipette Solution
11.3.3. Obtaining the Perforated Patch Configuration
11.3.3.1. Loading Patch Pipette
Fig. 11.1. Preparation and filling of patch pipettes. 1 Pull patch pipettes from capillary glass and polish using a microforge. 2 Place tip of patch pipette into a suspended droplet of amphotericin-free pipette solution for 5 s. 3 The antibiotic-free region should not extend more than 0.5 mm from the pipette aperture. 4 Back-fill patch pipette with amphotericin-containing pipette solution. 5 Ensure that no bubbles exist at the interface of the two solutions and proceed immediately to electrophysiology.
2. Load amphotericin-free pipette solution into a 1-ml plastic syringe and affix a filter disc.
3. Dispense pipette solution through the filter disc until a drop of filtered solution appears and is suspended from the filter disc.
4. Place the tip of the patch pipette into the drop of filtered pipette solution for 5 s (see Note 9).
5. Swirl the glass beaker or vial containing the amphotericin B pipette solution for several seconds (see Note 10).
6. Pierce the needle of the pipette-loading syringe through the foil and load the amphotericin-containing pipette solution. Repeat steps 4–6 each time a patch pipette is used (see Note 11).
7. Immediately, back-fill the amphotericin pipette solution into the patch pipette and proceed to the electrophysiology (see Note 12).
1. Place the patch pipette in the electrode holder.
2. Apply a very small amount of positive air pressure to the patch pipette via the side-port of the electrode holder (see Note 13).
3. Immerse the patch pipette into the bath and zero the offset potential (see Note 14) and Apply 1 mV test pulses from the 0 mV holding potential.
4. Select a single cell that does not make connections with neigh-bouring cells (see Note 15).
5. Position the patch pipette against the cell and apply negative pressure to the patch pipette to obtain a gigaohm (GΩ) seal (see Note 16).
6. Once a gigaohm seal has been obtained, change the test pulse to 10 mV, set the holding potential (usually −80 to −60 mV), and cancel the pipette capacitance using the amplifier fast capacitance cancellation settings.
7. Electrical access to the cell by perforation is indicated by the appearance of slow capacitance currents. The access resistance (Ra) decreases over time, as more amphotericin pores are formed in the membrane enclosed by the patch pipette. This can be observed by the change in the shape of the capacitance transients: from low amplitude that decays slowly (long time constant) to larger amplitude with a short decay time (Fig. 11.2 see also Note 17). Monitor access resistance using the whole-cell (slow) capacitance cancella-tion settings on the amplifier and update the settings every minute. Once the resistance is below an acceptable value (e.g. <10 MΩ), series-resistance compensation circuitry may be engaged if required, and the experiment can commence.
Access resistances that are 3–4 times the pipette resistance 11.3.3.2. Obtaining
a Gigaohm Seal and Perforation
Fig. 11.2. (A) Representation of a transient capacitance current evoked by a voltage step. (B) Capacitance currents recorded from a HEK293 cell undergoing perforation by amphotericin B in the patch pipette. Representative currents at the start of the recording (1), after 100 s (2), and after 200 s (3). The decrease in the access resistance is indicated by the increase in the amplitude of the current and the rate of decay (shorter time constant).
Fig. 11.3. Sample whole-cell perforated patch recordings from a HEK293 cell. Following perforation the whole-cell capacitance was cancelled and 80% series-resistance compensation was applied. (A) Representative currents were evoked by 100 ms pulses from −100 to +100 mV from a holding potential of −80 mV. The current–voltage relationship is shown in (B).
are typically obtained. A sample recording under these con-ditions is shown in Fig. 11.3.
1. Although filamented capillaries can be used, the lack of a fila-ment in the capillary glass gives better “tipping” partition-ing between amphotericin-containpartition-ing and amphotericin-free solutions (see Subheading 11.3.3.1).
2. The pipette solution is designed to closely match the intra-cellular concentrations of K+, Na+, and Cl−, which permeate amphotericin B pores. Whilst SO42− is used here, other suit-able impermesuit-able counteranions, such as aspartate, may also be used. The gradual exchange of ions between cytosol and patch pipette to reach the Donnan equilibrium results in a voltage drift over time.
3. Cells can be plated onto glass cover slips if preferred and coat-ing agents (e.g. poly-D-lysine) used if poorly adherent.
4. Do not attempt to mix with the pipette tip as the amphotericin B dissolved in DMSO will stick to the plastic tip. Flick the microtube to gather the contents to the bottom, dispense the DMSO along the side of the tube, and then sonicate.
5. The use of a glass container is recommended as the ampho-tericin–DMSO sticks to plastic.
6. This will appear as an apparently insoluble yellow “slug” at the bottom of the flask. Dissolve the “slug” by repeated pipetting with a 1 ml pipette (e.g. Gilson P1000).
7. The foil prevents destruction of the antibiotic by light.
8. Patch pipettes should be prepared as if they were to be used in standard whole-cell recording with relatively low resistances to reduce the membrane charging time constant and series-resistance errors. It is critical that patch pipettes are clean and polished to ensure optimal seal formation in as short a time possible, and it is worthwhile optimising the cell preparation and patch pipette fabrication.
9. The presence of amphotericin B in the pipette solution can prevent seal formation, and likewise any amphotericin B that leaks from the patch pipette near the cell can damage the membrane. By “tipping” the pipette tip with antibiotic-free solution, optimal seal formation should take place before the amphotericin reaches the tip where it invades the mem-brane. How much antibiotic-free solution should be loaded into the patch pipette tip (i.e. how long the patch pipette should