Diaphragm clamp operation experiment
Diaphragm clamp operation experiment
The membrane clamp technique can be applied to (1) characterization of membrane ion channels and (2) drug screening.
Operation method
Diaphragm clamp operation experiment
Principle
Membrane clamp technique is a method of contacting the cell membrane with a microslide electrode (diaphragm electrode or diaphragm pipette) and sealing it with an impedance of more than a gigaohm, so that a small area of the cell membrane (diaphragm) connected with the opening of the electrode tip is electrically separated from its surroundings, and the ionic currents of the ion channels in this diaphragm (pA level) are monitored and recorded by fixing a point on this basis. The core of the measurement loop is an I-V converter consisting of a field effect tube operational amplifier. When the positive and negative input terminals of the field effect tube operational amplifier are equipotential and the command potential is applied to the positive input terminal, the negative terminal and the diaphragm can be short-circuited to achieve the purpose of clamping equipotentially, and the shunt current between the tip of the diaphragm microelectrode and the silent piece forms a sealing connection of 10 GΩ or more is minimized, so that the current across the diaphragm can be measured as a recording current (lp) from the diaphragm electrode at 100 %. Measured.
Materials and Instruments
diaphragm clamp Move The study of membrane ion channel properties using membrane clamp is an arduous, meticulous and complicated work, which requires a high level of technical level and experimental conditions as a guarantee, and now we will roughly introduce the process of membrane clamp experiment, which roughly includes the following aspects.1. specimen preparation According to the different purposes of the study, different cell tissues can be used, such as cardiomyocytes, smooth muscle cells, tumor cells, etc., and now almost all kinds of cells can be used for membrane clamp study. For the cells used, the experimental requirements must be met, and the enzymatic isolation method is generally mostly used, and the cell culture method can also be used; in addition, due to the combination with molecular biology technology, molecular cloning technology is now also used to express different ion channels, such as the use of African clawed toad oocytes to express exogenous genes and so on.2. Electrode preparation Qualified membrane microelectrodes are the basic conditions for successful sealing of cell membranes. To successfully seal the cell membrane requires two factors to ensure, one is to try to cause a clean cell membrane surface, and the other is to make a qualified electrode. The first step is to choose the appropriate glass capillary, the material can be used soft glass (soda glass, calcium carbide glass) or hard glass (borosilicate glass, aluminum silica glass, quartz glass). Soft glass electrodes are commonly used for making whole-cell recordings, and hard glass is commonly used for ion single-channel recordings because of its low conductivity and low noise. Diaphragm microelectrodes are made by pulling glass capillary tubes with an electrode puller in three steps:The first step is divided into two pulling, the first time to pull the length of 7 ~ 10mm, the diameter of less than 200um, on this basis for the second pulling, and ultimately make the tip of the diameter of 1 ~ 2um, the two-step pulling is mainly to make the electrode front end of the taper becomes larger, the narrowness of the length of the shortening, so you can reduce the series resistance of the electrodes, but also reduce the electrode liquid dialysis time when the whole-cell recordings. Since the diaphragm microelectrode is most avoided to be contaminated with dust and dirt, and even more avoided to touch the parts near the tip, it is generally required to be made before use.The second step is to coat the front end of the electrode with a silicone resin (sylgard), which is intended to reduce the capacitance between the electrode and the perfusate and to form a hydrophilic interface. After this treatment, the above capacitance can be reduced from 6 to 8 pF to less than 1 pF. Silicone resin has no effect on the formation of seals of Giga, but reduces background noise, which is important for single-channel recordings. Satisfactory results can be obtained without the use of silicone resin when performing whole-cell recordings, and usually the microelectrodes are polished after applying silicone resin, but it is preferable to polish them within one hour after application, otherwise it is difficult to change the shape of the electrode tip.The third step is polishing, the electrode is fixed on the microscope table, the tip is close to the heating wire under the microscope, when energized and heated, the tip of the electrode can be seen to retract slightly, at this time, the electrode becomes smooth, and the tip of the impurities burned away, to get a cleaner surface. This is conducive to close sealing with the cell membrane, and easier to maintain stability after sealing.The electrode should be filled with electrode liquid before the experiment, because the electrode tip is thin, so before filling, the liquid inside the electrode should be filtered with 0.2 um filter membrane. General electrode filling can be divided into filling tip (tipfilling) and backfilling (backfilling) two steps. The tip of the electrode is immersed in the inner liquid for 5s, due to capillary action of the solution will enter the tip of the electrode, and then from the back end of the electrode with a small polypropylene injection tube inserted near the tip of the solution filled to 1/4 length, with a finger gently popping off the tip of the residual air bubbles can be. The electrode resistance after filling is generally 2 to 5 MΩ;, while whole-cell recording is preferably 2 to 3 MΩ.3. Diaphragm clamp experimental system According to different electrophysiological experimental requirements, different experimental systems can be formed, but there are a number of common basic components, including mechanical parts (anti-vibration bench, shielding, instrumentation racks), optical parts (microscopes, video monitors, monochromatic light systems), electronic components (diaphragm clamp amplifiers, stimulators, equipment for data acquisition, computer systems) and micromanipulators (Figure 1). ). Common Problems Diaphragm clamp experiments are difficult and technically demanding, it is not difficult to master the relevant techniques and methods, but it is not so easy to process and analyze a large amount of experimental data to draw meaningful and valuable results and conclusions, there are many issues that need to be paid attention to and considered, including the reduction of noise, to avoid contamination of the electrode front, to improve the success rate of sealing, and in the specific experimental process, it is necessary to There are many more complicated issues to be considered, including how to select the recording mode, how to choose the electrode internal and external fluids for the recording of specific ionic currents, how to select the blocker and agonist, and how to carry out the correct data acquisition, etc., which need to be explored and solved continuously in scientific research practice. For more product details, please visit Aladdin Scientific website.
Figure 1
In most membrane clamp experiments, good mechanical stability of all experimental apparatus and equipment is required so that the relative motion between the microelectrode and the cell membrane is as small as possible. A vibration-resistant bench holds the inverted microscope and the micromanipulator fixedly attached to it, with the other equipment placed outside the bench. A shield made of copper wire mesh is grounded to prevent interference with the probe circuitry of the diaphragm clamp amplifier by stray electric fields from the surrounding environment. Instrument and equipment racks should be close to the bench to facilitate the mating of measuring instruments with optical instruments.The inverted microscope is the main optical component of the membrane clamp experimental system, which not only has a better visual effect and facilitates the contact of the glass electrode with the top of the cell, but also realizes focusing with the help of the moving objective lens and has a better mechanical stability. The video monitor is mainly used to monitor the operation during the experiment, especially to be able to correspond the sealing parameters (e.g. sealing impedance) to the morphology of the cells for good sealing.The membrane clamp amplifier is the core of the whole experimental system, which can be used for single-channel or whole-cell recording, and its working mode can be either voltage clamp or current clamp. In principle, the probe circuit of the membrane clamp amplifier, i.e., the I-V converter, has two basic structural forms, i.e., resistive feedback and capacitive feedback; the former is a typical structure, and the latter is particularly suitable for ultra-low-noise single-channel recordings because of the use of feedback capacitors instead of feedback resistors, which reduces the noise. As for the membrane clamp experiment of special computer hardware and the corresponding software programs appear one after another, so that the membrane clamp experiment easy to operate, improve efficiency. Such as with the EPC-9-type membrane clamp amplifier (containing ITC-16 data acquisition/interface card) supporting the use of software PULSE/PULSEFIT, which can generate stimulus waveforms, control data acquisition, but also analyze the data, and at the same time has a lock-in amplifier for membrane capacitance monitoring, a variety of software functions integrated into one.4. Conducting Experiments, Recording and Analyzing Data The preparations are ready for experimental operations, data recording and analysis. The main focus here is on the formation of the high-resistance blocking (Fig. 2).
To the electrode continuously apply a 1mV, 10 ~ 50 ms step pulse stimulation, electrode into the water resistance of about 4 ~ 6MΩ;, at this time in the computer screen display box can be seen in the current waveform generated by the test pulse. At the beginning of the gain should not be set too high, generally can be 1 ~ 5mV/pA, so as not to saturate the amplifier. Due to the difference in ionic composition between the extracellular fluid and the electrode internal fluid caused by the liquid junction potential, so the general electrode just into the water when the test waveform baseline is not on the zero line, must first hold the voltage is set to 0mV, and adjust the electrode misalignment control so that the electrode DC current close to zero. Using a micromanipulator to bring the electrode close to the cell, when the tip of the electrode is in contact with the cell membrane the sealing resistance indication Rm will rise, and when the electrode is pressed slightly downward, the Rm indication will rise further. When a slight negative pressure is applied to the electrode through a thin plastic tube and the cell membrane is well characterized, Rm will generally rise rapidly within 1 min until a high impedance seal of GΩ level is formed. Generally when Rm reaches about 100MΩ, a slight negative voltage (-30 to -10mV) applied to the electrode tip helps the formation of GΩ seal. The phenomenon at this point is that the current waveform becomes flat again, causing the electrode to hyperpolarize from -40 to -90mV, which helps to accelerate the formation of the seal. To confirm the formation of the GΩ seal, the gain of the amplifier can be increased so that the current waveform can be observed to remain flat except for the capacitive tip currents that appear at the beginning and end of the pulse voltage.After the formation of the high-impedance seals and before recording the experimental results, the parameters are usually compensated according to the requirements of the experiment in order to obtain realistic results. It should be noted that the bandwidth of the amplifier should be set appropriately, e.g., 10 kHz, so that no useless information beyond this bandwidth will be observed at the current monitoring end.Diaphragm clamp experiments are difficult and technically demanding, to master the relevant techniques and methods is not very difficult, but from a large batch of experimental data, after processing and analyzing, to derive meaningful and valuable results and conclusions, it is not so easy, there are many need to pay attention to and consider the issues, including reducing noise, avoiding contamination of the electrode front end, improve the success rate of the sealing of the specific experimental process, but also need to There are many more complicated issues to be considered, including how to select the recording mode, how to choose the electrode internal and external fluids for the recording of specific ionic currents, how to select the blocker and agonist, and how to carry out the correct data acquisition, etc., which need to be explored and solved continuously in scientific research practice.