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Patch Clamp Protocol
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Mary Johnson (han at labome dot com) Synatom Research, Princeton, New Jersey, United States DOI //dx.doi.org/10.13070/mm.en.1.190 Date last modified : 2017-06-03; original version : 2011-09-01 Cite as MATER METHODS 2011;1:190
Abstract
A detailed step-by-step description of the standard patch clamp protocol and Labome survey results for vibratomes and patch-clamp amplifiers. related topics method
Introduction 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 includes current clamp and 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.
Reagent Setup
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. s CRITICAL: The osmolarity should be between 305 and 315 mosm. Mix well and bubble in 95% O2–5%CO2. Microelectrode solution
(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.). s CRITICAL: An osmolarity between 260 and 280 mosm works best. Filter the solution at 0.2 µm and storing at 4°C. Equipment Setup
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
Table 1. Patch clamp equipments. Note: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.
Procedure 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 M. s 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. s 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. s CRITICAL STEP 9. Obtain a G seal spontaneously. If not, briefly apply light suction by mouth until the resistance reaches at least 1 G. 10. Once a G seal has been formed, proceed to obtain the desired patch-clamp configuration. 1. Cell-attached configuration: Upon acquiring a G seal one can proceed with the experiment. In this configuration, the microelectrode solution should resemble extracellular medium. 2. Inside-out configuration: After obtaining a G 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 G seal. The microelectrode solution should resemble 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. s 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 G 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 formation of an excised membrane ‘bleb.’ 11. Analyze recordings. Most acquisition software comes bundled with analysis software.
Vibratomes and patch clamp amplifiers in literature Supplier
Num References
Leica
8
[1-8]
Dosaka
2
[2, 9]
Campden Instrument 1
Labome surveys formal publications for reagents and instruments. Table 2 lists the two suppliers of vibratomes cited in the 11 publications indicating vibratomes. Table 3 lists the major suppliers of patch clamp amplifiers from the surveyed publications.
[10]
Table 2. Major suppliers of vibratomes.
Troubleshooting
Supplier
Main model
Num References
Molecular Devices / Axon Instruments AxoPatch 200B, Digidata 1322A, Digidata 1440A, MultiClamp 700B 35
[1-5, 11-40]
Heka Elektronik
5
[41-45]
Dagan Corporation
2
[46, 47]
Warner
2
[16, 38]
EPC-10
Table 3. Major suppliers of patch clamp amplifiers in the publications. Suppliers such as Hugo Sachs Elektroniks, Piezosystem Jena, A-M Systems, Neuralynx and others have one citation each. I cannot obtain G seals 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 M for mature neurons, 8–12 M 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. obtain a G seal but cannot “break in” because I lose the seal when trying 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. My seals do not last very long 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. I am not sure whether I am patching the right cell type 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. I am not sure whether my preparation is healthy 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). Does age of the animal matter?
Yes. up to P12, it is durable. After P12, it becomes increasingly difficult. My electrode measures 7M in the saline, but as soon as I exert pressure, it goes up to 15M. 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 >50M 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. 4-6M) and start deeper in the brain (say, layer 2/3). What is the ideal tip resistance?
It depends, but just to patch and establish current clamp recording, 5M 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 3M. 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 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. I cannot exert pressure / suction. Why?
Check your Tee junction. How much biocytin should I put in my internal solution?
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