Patch Clamp Assay - A detailed step-by-step description of the standard patch clamp protocol

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.

1. Patch Clamp Assay https://www.creative-bioarray.com/

2. Page 1/10 Introduction 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 of potential drugs, especially cardiotoxicity. It includes a current clamp and a voltage clamp, as well as 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.

3. Page 1/10 Fiber-optic array scanning technology (FAST) Cell-attached Patch Outside-out Excised Patch Inside-out Excised Patch Perforated Patch Whole-cell Patch Types of Patch Configurations

4. Page 1/10 Cell-attached Patch In this configuration, the patch pipette forms a highly resistant seal with the membrane, called  gigaseal , enabling pico scale measurement of the membrane potential. The cell-attached patch configuration does not require the electrode to penetrate the membrane, leaving the cell intact during patch clamp. Considered the original patch clamp, the cell-attached patch clamp measures membrane potential from the electric current that passes an ion channel.

5. Page 1/10 Whole-cell Patch The disrupted membrane allowed the pipette buffer to directly contact the cytoplasm, resulting in membrane potential measurement, or an instant current injection that compensates for the voltage difference between the intracellular and extracellular environment. This type of patch clamp is most similar to intracellular recording and is used for the overall assessment of ion channels distributed in the whole cell or tissue slices. In this configuration, the tissue or cells are immersed in the bath solution together with the bath electrode. To obtain the membrane potential, puncture the cell membrane with the patch pipette.

6. Page 1/10 Outside-out Excised Patch Modified from a whole-cell patch clamp, the outside-out excised configuration measures an individual ion channel by rupturing the cell membrane. Here, the patch pipette is pulled away after it makes contact with the membrane, causing the membrane phospholipids to aggregate around the patch pipette. This lets users test the effect of extracellular factors directly on the ion channel.

7. Page 1/10 Inside-out Excised Patch The inside-out excised patch configuration necessitates a bath solution that resembles the cytosol to prevent cytosolic washout, making this patch clamp type suitable for testing the influence of a specific extracellular factor on the ion channels. Unlike the outside-out type, the patch pipette is briefly removed from the bath solution, leaving the patch with the bath-facing cytosolic side.

8. Page 1/10 Perforated Patch Another whole-cell patch modification, a perforated patch clamp records the whole-cell membrane potential. The setup in the perforated patch is like the whole-cell patch clamp, except an antifungal such as amphotericin B and nystatin is added to the pipette solution. It acts as a membrane-perforating agent that only allows small ions to pass through the punctured membrane, blocking the loss of large cytosolic components.

9. Page 1/10 125 mM NaCl, 2.5 mM KCl , 1 mM MgCl 2 , 2 mM CaCl 2 , 1.25 mM NaH 2 PO 4 , 25 mM NaHCO 3 and 25 mM glucose, pH 7.4. ▲ Critical: The osmolarity should be between 305 and 315 mosm . Mix well and bubble in 95% O 2 -5%CO 2 . Artificial cerebrospinal fluid (ACSF) (For whole-cell recordings: 130 mM KCl , 5 mM NaCl, 0.4 mM CaCl 2 , 1 mM MgCl 2 , 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℃. Microelectrode solution Patch Clamp Protocol

10. Page 1/10 Platinum wire U-piece with nylon threads Inverted suction pipette Syringes Microscope with fiber optic light source Computer with monitor and software Patch clamp amplifier Vibration isolation stage Microelectrodes Microelectrode holder Micromanipulator Recording chamber Drug application system Electrode puller (Sutter P-1000 multi-step puller) Table 1. Patch clamp equipment setup. Note: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. Patch Clamp Protocol

11. Page 1/10 01 03 Acute brain slices/cultured 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. 02 Pull recording microelectrodes to an input resistance of 5-8 MΩ. (▲Critical step) Set the bath application system to run at 1-2 ml per minute. Place the slice/cells in. 04 06 Fill the recording microelectrode with electrode solution. 05 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. Place the microelectrode in the pipette holder. Apply positive pressure using a 10-ml syringe by displacing the plunger about 1 ml. (▲Critical step) Patch Clamp Protocol

12. Page 1/10 Patch Clamp Protocol 07 09 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. 08 Once an obvious and steady change in microelectrode resistance is obtained, release the positive pressure rapidly. (▲Critical step) 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. 11 Analyze recordings. Most acquisition software comes bundled with analysis software.

13. Page 1/10 Troubleshooting Problem Solution I cannot obtain GΩ seals Make sure your preparation is healthy and has always been oxygenated; check the pH and osmolarity of your ACSF and filling solution. Make sure you are placing the electrode in an area of high cell density. 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). Check the pressure line to the microelectrode holder for leaks. Clean the microcapillaries and make sure that you are not contaminating them with oils while handling. Obtain a GΩ seal but cannot “break in” because I lose the seal when trying Check the pressure line to the microelectrode holder for leaks or obstructions. Try the “zap” function in your amplifier. Try a different microelectrode. Lowering the resistance may help.

14. Page 1/10 Troubleshooting Problem Solution My seals do not last very long Once in whole-cell mode, apply light positive pressure. Check the shape of your microelectrode tip. Make sure your preparation is healthy. Check the osmolarity of the solutions. Make sure the ACSF bath and drug application system does not have air bubbles. Make sure no vibration of the stage or microelectrode holder. I am not sure whether I am patching the right cell type Make sure your preparation is healthy. Be familiar with the basic electrical properties of your cell of interest. Include a dye to track cell morphology post hoc. I am not sure whether my preparation is healthy Perform a rapid cell-death and survival assay using representative preparations and the fluorescent markers propidium iodide (dead cells) and Syto-11 (live cells).

15. Page 1/10 Troubleshooting Problem Solution Does the age of the animal matter? Yes. up to P12, it is durable. After P12, it becomes increasingly difficult. My electrode measures 7MΩ in the saline, but as soon as I exert pressure, it goes up to 15MΩ. Should I patch? No. Your solution contains dust which clogs the tip of the electrode. If this happens 3 times repeatedly, refilter your pipette solution. My electrode is >50MΩ with visible capacitance!! help! You have bubbles in the tip of your pipette. Take the pipette out and give it a few taps. Does penetration angle matter? Probably Yes. In general, 45 degree or shallower makes better giga seals. I always get dendrites, but my boss needs somatic recording. Make a bigger patch electrode ( e.g. , 4-6MΩ) and start deeper in the brain.

16. Page 1/10 Troubleshooting Problem Solution What is the ideal tip resistance? 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. How do I chloride my silver electrode? Put it in bleach for 15-30 min. Electrolysis in saline (150-300 mM NaCl) with 5-10V. Voltage shows slow DC shift, especially when I turn on step command. What should I do? It is likely that your electrode (either reference or electrode) is not chlorided . ( i.e. , silver is exposed, instead of AgCl, or silver is somehow oxidized). Put the silver wires in bleach for 15 min or so. I cannot exert pressure/suction. Why? Check your Tee junction. How much biocytin should I put in my internal solution? 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.

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