The patch clamp is a laboratory technique in electrophysiology that allows investigation of the electrical excitability of neurons and the functional properties and densities of ion channels. It is widely used to evaluate the toxicity, especially cardiotoxicity, of potential drugs, for example, [1]. It includes a current clamp and a voltage clamp, and several patch configurations (whole cell, single channel, perforated patch, etc.) varied with respect to membrane integrity, membrane orientation, and continuity between the intracellular space and intrapipette solutions. Here is a standard protocol of blind patch clamp. Automated patch clamp platforms have been developed in recent years [2, 3].

Anesthetics, experimental animals, vibratome, dissection dish, and other tools for dissection and slice handling.

125 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃ and 25 mM glucose, pH 7.4.
▲ CRITICAL: The osmolarity should be between 305 and 315 mosm. Mix well and bubble in 95% O₂–5%CO₂.

(For whole-cell recordings: 130 mM KCl, 5 mM NaCl, 0.4 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES and 11 mM EGTA, pH 7.3.).
▲ CRITICAL: An osmolarity between 260 and 280 mosm works best. Filter the solution at 0.2 μm and store it at 4°C.

1. Acute brain slices / cultured cells / enzymatically isolated cells should be superfused in ACSF / extracellular solution and continuously gassed with carbogen (5% CO₂/95% O₂) for at least 2 h at room temperature before recording.
2. Pull recording microelectrodes to an input resistance of 5–8 MΩ.
   ▲ CRITICAL STEP
3. Set the bath application system to run at 1–2 ml per minute. Place the slice/cells in.
4. Fill the recording microelectrode with electrode solution.
5. If documenting cellular morphology post hoc is desired, include the intracellular dye filling of choice in the micropipette solution. Available dyes include Lucifer yellow, Cell Tracker, biocytin, Alexa biocytin, neurobiotin, etc.
6. Place the microelectrode in the pipette holder. Apply positive pressure using a 10-ml syringe by displacing the plunger about 1 ml.
   ▲ CRITICAL STEP
7. Set the amplifier to voltage clamp and apply a test pulse of 5–10 msec and 20 mV amplitude. Slowly approach the area of interest until there is an obvious change in the test pulse amplitude.
8. Once an obvious and steady change in microelectrode resistance is obtained, release the positive pressure rapidly.
   ▲ CRITICAL STEP
9. Obtain a GΩ seal spontaneously. If not, briefly apply light suction by mouth until the resistance reaches at least 1 GΩ.
10. Once a GΩ seal has been formed, proceed to obtain the desired patch-clamp configuration.
    1. Cell-attached configuration: Upon acquiring a GΩ seal one can proceed with the experiment. In this configuration, the microelectrode solution should resemble the extracellular medium.
    2. Inside-out configuration: After obtaining a GΩ seal, slowly pull the pipette away from the cell. Eventually, a small piece of cell membrane will be detached from the cell surface without losing the GΩ seal. The microelectrode solution should resemble the extracellular medium in this configuration also.
    3. Whole-cell configuration:
       1. In this configuration, the microelectrode solution should approximate intracellular ionic composition.
       2. To record in whole-cell mode, change the voltage clamp to a negative voltage close to the cell resting potential (–60 mV for radial glial cells) and correct for fast capacitance. Apply continuous light suction by mouth until the membrane breaks as evidenced by a change in the capacitance and the test pulse current. ▲ CRITICAL STEP
       3. If doing perforated patch recordings, front-fill the microelectrode with electrode solution without antibiotic, back-fill with electrode solution containing antibiotic. After a GΩ seal is obtained, simply set the voltage clamp near the resting potential and wait for the resistance to slowly decrease and stabilize.
    4. Outside-out configuration: After obtaining a whole-cell recording, very slowly withdraw the pipette until resistance increases greatly, indicating the formation of an excised membrane ‘bleb.’
11. Analyze recordings. Most acquisition software comes bundled with analysis software.

Labome surveys formal publications for reagents and instruments. Table 2 lists the suppliers of vibratomes cited in the formal publications indicating vibratomes. Table 3 lists the major suppliers of patch clamp amplifiers from the surveyed publications. Patch clamp amplifiers like Axopatch 200B are also used in nanopore experiments [9]. For example, Yang J et al recorded the whole-cell currents in HEK293 cells with MultiClamp 700B amplifier and 1550B digitizer from Molecular Devices [4]. Gong J et al recorded glutamate-gated currents of wild-type and various variants of mouse GluK2 as well as GLR-3 expressed in CHO cells a Multiclamp 700B amplifier [10].

1. Make sure your preparation is healthy and has always been oxygenated; check the pH and osmolarity of your ACSF and filling solution.
2. Make sure you are placing the electrode in an area of high cell density.
3. Check the shape of your microelectrode tip. Keep the resistance in the right range (4–6 MΩ for mature neurons, 8–12 MΩ for small cells).
4. Check the pressure line to the microelectrode holder for leaks.
5. Clean the microcapillaries and make sure that you are not contaminating them with oils while handling.

1. Check the pressure line to the microelectrode holder for leaks or obstructions.
2. Try the “zap” function in your amplifier.
3. Try a different microelectrode. Lowering the resistance may help.

1. Once in whole-cell mode, apply light positive pressure.
2. Check the shape of your microelectrode tip.
3. Make sure your preparation is healthy.
4. Check the osmolarity of the solutions.
5. Make sure the ACSF bath and drug application system does not have air bubbles> Make sure no vibration of the stage or microelectrode holder.

1. Make sure your preparation is healthy.
2. Be familiar with the basic electrical properties of your cell of interest.
3. Include a dye to track cell morphology post hoc.

1. Perform a rapid cell-death and survival assay using representative preparations and the fluorescent markers propidium iodide (dead cells) and Syto-11 (live cells).

Yes. up to P12, it is durable. After P12, it becomes increasingly difficult.

No. Your solution contains dust which clogs the tip of the electrode. If this happens 3 times repeatedly, refilter your pipette solution.

You have bubbles in the tip of your pipette. Take the pipette out and give it a few taps.

Probably Yes. In general, 45 degree or shallower makes better giga seals.

Make a bigger patch electrode (e.g., 4-6MΩ) and start deeper in the brain (say, layer 2/3).

It depends, but just to patch and establish current clamp recording, 5MΩ is probably the choice. Smaller (i.e., higher resistance) is slightly easier to form a seal, but more difficult to break in, and the access resistance will be bigger. Bigger (i.e., lower resistance) will get you to soma and probably good voltage clamp, but seal formation will be more difficult. I’ve never managed to make a seal with electrodes smaller than 3MΩ.

Put it in bleach for 15 – 30 min.

Electrolysis in saline (150 – 300 mM NaCl) with 5 – 10V.

It is likely that your electrode (either reference or electrode) is not chrolided. (i.e., silver is exposed, instead of AgCl, or silver is somehow oxidized). Put the silver wires in bleach for 15 min or so.

Check your Tee junction.

Again, the answer is that it depends. Usually, 0.5% to 2% (w/v) is more than enough. Mind the osmolality. It should be around 290-310.