Advan. Physiol. Edu. 32: 209-211, 2008;
doi:10.1152/advan.90138.2008
1043-4046/08 $8.00
ADV PHYSIOL EDUC 32:209-211, 2008
© 2008 American Physiological Society
HOW WE TEACH
A reliable whole cell clamp technique
Chenhong Li
Key Laboratory of Molecular Biophysics of Ministry Education of China, Institute of Biophysics and Biochemistry, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
Address for reprint requests and other correspondence: C. Li, Key Laboratory of Molecular Biophysics of Ministry Education of China, Institute of Biophysics and Biochemistry, College of Life Science and Technology, Huazhong Univ. of Science and Technology, Wuhan 430074, China (e-mail: lichhhust{at}yahoo.com)
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Abstract
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This article describes a simple whole cell formation technique that the author invented in teaching and experiments. The implementation of the invented technique is a syringe with a hole and slot. With the newly invented technique, novices will shorten their learning curve and veterans will increase their success rate. The invented technique lightens the labor of the experimenter and improves the success rate and quality of whole cell preparations. The article also provides an idea to design an automated whole cell formation implementation. The tools developed in this technique make the patch-clamp experiment easy to teach and learn.
Key words: electrophysiological technique; patch clamp; whole cell configuration
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Introduction
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SINCE THE PATCH-CLAMP RECORDING TECHNIQUE was invented by Neher and Sakamann (4) in 1976, the technique has been widely used to study the physiological function and pathological mechanisms of organisms in vivo and vitro at molecular levels. Even though the technique is well known by now, the operation is still one of the most difficult tasks to perform. The technique comes with a big drawback: low throughput and high labor cost. However, since there is no better substitute for this technique so far, it will remain to be one of the most important tools for advanced life science. Many companies have started to develop an automated patch clamp. However, the automated patch clamps developed so far have not been truly used in basic life science research (6). The success of patch-clamp applications greatly depends on the experience and technical skills of people. These experiences and skills are built in people's minds and hands. It is not easy to quantify them. This greatly limits the development of automated patch clamps. Therefore, the manual patch clamp is still the main methods in life cell research, such as ion channel drug screening (6).
There are several patch-clamp modes (2, 3, 5). The whole cell mode is the basic mode. It is also the most difficult mode to achieve. The whole cell mode requires making a hole on a patch of cell membrane at the tip of the pipette that attaches to the cell. Furthermore, the seal resistance between the pipette and cell needs to be above gigaohms to form a so-called gigaseal (2). If the seal resistance is not high enough, too much electric current will be leaked, and the whole cell clamp will become useless. There are several techniques to make a hole on the patch (1, 2), such as by mouth suction, electric pulse, syringe, and negative pressure pump. The success of mouth suction completely depends on the individual's experience and skill, which are very hard to quantify. The later techniques are quantitatively defined. However, their success rates are still very low.
In this article, a simpler, easier, and more reliable laboratory technique of making a whole cell clamp is described. The technique is based on the syringe and negative pressure pump method. A mechanism was invented to quantitatively control the process. With this control mechanism, the whole cell clamp-making process becomes more robust. A novice experimenter can shorten the learning curve, and an experienced veteran can increase their success rate. Shortening the learning curve is also needed for a veteran experimenter because after a long period of interruption, the veteran still needs time to get back his/her feeling and experience.
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MATERIALS AND METHODS
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Tool Modifications
As shown in Fig. 1, the tools used in this experiment are a syringe, control valve, gel tube (external diameter: 3 mm, inside diameter: 1 mm), and syringe needle (external diameter: 1.6 mm, with the tip cut off) connected in sequence. The tools are just like those used in any syringe and negative pressure pump method. However, the difference is the syringe. A hole and slot were made on the two ends of the 1-ml syringe, respectively. As shown in Fig. 1, hole A is at the 0.1-ml position with a diameter of 4 mm. Rectangular slot B is between the 0.9- and 1-ml positions with a size of 3 x 6 mm. Besides the tools shown in Fig. 1, a glass pipette, patch-clamp instrument, and inverted microscope are also needed in the experiment.
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Fig. 1. Tools used in the whole cell technique. Top: syringe with hole A and slot B connected to a control valve and gel tube. The gel tube is connected to a pipette (not shown). Bottom: zoomed in pictures of the two openings [hole A (left) and slot B (right)].
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Operation of the Whole Cell Clamp Tool
The following steps describe how to operate the whole cell clamp tool.
1. Applying positive pressure to the pipette.
Push the plunger to the bottom of the syringe, which produces a positive pressure corresponding to a 0.1-ml air column. Then close the control valve to keep the positive pressure. Now, pull the plunger out beyond hole A so that the positive pressure will be released through hole A when the control valve is open later.
2. Releasing the positive pressure in pipette.
When the tip of the glass pipette touches the cell surface and the responding current is reduced to a desired level, i.e., 1/2--1/3 of the origin peak current in most cases, open the control valve to release the positive pressure through hole A.
3. Generating negative pressure in the pipette.
Place the forefinger of the left hand on hole A to seal the hole. At the same time, pull the plunger out for 
0.2--0.6 ml with the right hand to generate a negative pressure in the pipette. Then close the control valve to keep the negative pressure, and release the forefinger from hole A. When the seal resistance reaches gigaohms, open the control valve to release the negative pressure to keep the seal stable. Of course, sometimes the seal resistance doesn't reach gigaohms; releasing the negative pressure will also help to form the gigaseal. In this way, a cell-attached mode is formed (Fig. 2A).
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Fig. 2. Whole cell mode forming procedure with two separated negative pressures (model pictures). A: the cell-attached mode was formed with gigaohm seal resistance. B: after the cell-attached formation, the first negative pressure sucked the patch of membrane into the pipette further and created a thinning spot. C: the second negative pressure that was quickly created and quickly released generated a busting power (inside the cell) that cracked the patch and formed the whole cell mode.
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4. Making a hole in the patch of the cell membrane.
Close the control valve and then adjust the distance between the plunger and slot B. (Alternatively, the syringe may be separated from the control valve to adjust the position of the plunger and then reconnected to the control valve.) Normally, the plunger can be placed 0.4--0.6 ml away from slot B. Seal hole A with the left hand forefinger, open the control valve, and pull the plunger out for 0.1--0.2 ml with the right hand to generate a negative pressure in the pipette (Fig. 2B). Allow the negative pressure to stabilize for a while, and then pull the plunger out over slot B quickly. The negative pressure is formed quickly and released quickly. In this process, a patch of the cell membrane at the tip of the pipette will crack and form a hole. Consequently, a large transient capacitance suddenly appears. At the same time, the high seal resistance is maintained. In this way, a whole cell mode is formed (Fig. 2C).
Solutions
The standard intracellular solution used consisted of (in mM) 6 NaCl, 130 K-aspartate, 2 MgCl2, 5 CaCl2, 11 EGTA, and 10 HEPES, with pH adjusted to 7.2 by KOH. The extracellular solution consisted of (in mM) 130 NaCl, 2 MgCl2, 5 HEPES, 5.6 KCl, and 1.8 CaCl2, with pH adjusted to 7.4 by NaOH.
Data Analysis
Data were analyzed using GraphPad 4.02 software. Data are presented as means ± SE. The significance was tested by an unpaired Student's t-test. Differences in mean values were considered significant at a probability of P 
0.05.
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RESULTS
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Access resistance is an important measure of the quality of whole cell formation. Figure 3A shows a histogram of access resistances of 22 cells (HEK-293 cells) sealed using this new technique. Figure 3B shows a histogram of access resistances of 20 cells (HEK-293 cells) sealed using the transitional technique. The results shown in Fig. 3, A and B, indicate that the access resistances in both groups are Guassian normal distributions. However, their mean values were 3.755 ± 0.132 and 5.476 ± 0.189 M
, respectively. The significance was tested by an unpaired Student's t-test. The differences in mean values were indeed significant (P 0.01). Obviously, the new technique significantly improved the quality of whole cell formation compared with the transitional technique.
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Fig. 3. A and B: histograms of access resistances. A: access resistances of 22 cells sealed with the new technique. The solid line is the fitted Gaussian function. B: access resistances of 20 cells sealed with the traditional technique. The two histograms are not shown to compare success rates.
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The success rates of whole cell formation are very different depending on individual experiences. To compare the success rates of the techniques only and exclude the effects of experience, four novices were chosen to conduct the experiment of whole cell formation using the new technique and the transitional technique (mouth suction technique). To reduce the artificial deviation, the two techniques were used in alternation. Two novices used the new technique first and then the traditional technique second, whereas the other two novices used the transitional technique first and the new technique second. Novices sealed 30 cells (HEK-293 cells) by the traditional technique, but no seals were obtained. When they used the new technique for another 30 cells (HEK-293 cells), the first successful formation of the whole cell mode happened between the 8th cell and 12th cell, and then the seal success rate gradually increased. For the 30 cells, the average success rate was 32.48 ± 2.86% using the new technique compared with 0% by the traditional technique. It is obvious that the new technique shortened the learning curve for the novices.
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DISCUSSION
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This technique produces a high-quality seal. In addition, it reduces the frustration of the experimenter and improves success rates in whole cell mode experiments.
In the modified syringe, hole A has several additional advantages. First, when connecting the syringe with the control valve, the existence of hole A ensures that no positive pressure will be generated inside the pipette. A little positive pressure would destroy the high sealing resistance that was formed. The unwanted positive pressure was the main reason of failures when the traditional syringe method was used.
Second, as shown in Fig. 2B, after the cell-attached mode is formed, the patch of the cell membrane at the tip of the pipette may sometimes be cracked to form a whole cell model by the first negative pressure before going to the next step. In this case, hole A can be used to promptly release the negative pressure by taking off the left forefinger. If the negative pressure is not promptly released, the whole cell mode is broken right away. This was another reason for the failures that contributed to the low rates of success of the traditional method.
The main function of slot B on the modified syringe is to produce a controlled bursting power. When the plunger is quickly pulled out, a sucking force is formed quickly. However, the sucking force disappears in a controlled manner when the plunger reaches slot B. This is equivalent to producing a bursting power inside the cell. It is the same bursting power generated by mouth suction. As well known, the bursting power is a necessary condition to form the whole cell mode. Slot B makes the bursting power more controllable.
Of course, the technique is not truly quantified yet. However, it gives the experimenter an intuitive measurement of positive pressure and negative pressure. Therefore, the experimenter can make adjustments to control the timing and magnitude of the pressures. Obviously, this technique is much easier than using mouth suction, and the results are of much higher quality than those using negative pressure pumps.
In addition, the separated two-step negative pressures after the cell-attached mode greatly increase the success rate and improve the quality.
The new technique has been used in several types of cells [dorsal root ganglion (DRG), HEK-293, Ins-1, smooth muscle, and PC12 cells] in our laboratory. It was found that, although the technique fitted all cells, the DRG cells were easier to use to form the high-quality whole cell mode. In the future, more studies on cell types will be conducted using the newly improved technology.
In conclusion, the newly developed whole cell clamp technique reduces the labor, increases the success rate, and improves the quality of the whole cell formation. It also has good applications in education. The tools developed in this technique make the patch-clamp experiment easy to teach and learn.
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GRANTS
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This work was supported by National Nature Science Foundation of China Grant 30470646, Nature Science Foundation of Huazhong University of Science and Technology Grant 20071986, and Provincial Nature Science Foundation of Hubei Grant 2003ABA096.
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Acknowledgments
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The author thanks Prof. Susan Crowell for the help in editing this manuscript.
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REFERENCES
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- Axon Instruments. Axopatch 200B Patch-Clamp Theory and Operation, Axopatch 200B Manual. Sunnyvale, CA: Axon Instruments, chapt. 3, p. 35.
- Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[CrossRef][Web of Science][Medline]
- Hille B. Ion Channels of Excitable Membranes (3rd ed.). Sunderland, MA: Sinauer, 2001, p. 88.
- Neher E, Sakmann B. Channel currents recorded from membrane of denervated frog muscle fibers. Nature 260: 799–802, 1976.[CrossRef][Web of Science][Medline]
- Sakmman B, Neher E. Single-Channel Recording (2nd ed.). New York: Plenum, 1995, p. 8–10.
- Xu J, Wang X, Ensign B, Li M, Wu L, Guia Xu J A. Ion-channel assay technologies: que vadis. Drug Discov Today 6: 1278–1287, 2001.[CrossRef][Web of Science][Medline]
Copyright © 2008 by the American Physiological Society.
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Abstract
Introduction
