Inspection items based on patch-clamp technology
1. Technology Introduction
Membrane clamp technology is known as the "gold standard" for the study of ion channels. It is currently the most important technique for studying cell membrane currents and ion channels. Technically speaking, patch clamp is a microelectrode technique that uses a clamp voltage or current (usually a clamp voltage) to record the electrical activity of ion channels in cell membranes. In the early days of single-channel recording, a "patch", i.e., a piece of cell membrane, was a small piece of cell membrane that could contain one or several ion channels that was adsorbed and recorded by a glass microelectrode port. Later, the membrane clamp technology developed a variety of recording configurations, "membrane" can refer to a variety of single-channel recording configurations in the small membrane, but also can refer to the whole cell membrane in the whole-cell recording configuration to be detected. "Clamp", that is, the meaning of human control, according to the different electrical parameters of human control, membrane clamp technology can be divided into voltage clamp and current clamp two working modes. Voltage clamp (VC), that is, artificial control of the voltage difference between the two sides of the cell membrane (i.e., transmembrane potential), in this condition to detect the transmembrane current, the specific clamp pressure requirements depend on the characteristics of the ion channel. Current clamp (IC), on the other hand, is to artificially control the current applied to the cell membrane and observe the transmembrane potential under these conditions. Voltage clamp is a common mode of operation in membrane clamp experiments, which can better study the voltage dependence of the channel and easily distinguish the electrical response caused by other stimuli and membrane potential changes, and the results recorded in the voltage clamp state are easy to analyze.
Fig. 1 Patch clamp system
2. Principle of diaphragm clamp technique
The principle of the diaphragm clamp technique is: a glass microelectrode with an end diameter of 1.5-3.0 um is used to contact the surface of the cell membrane, and then a gigohm-level impedance seal is formed between the tip and the cell membrane by negative pressure attraction. The cell membrane area inside the tip mouth forms an electrical separation from other surrounding areas, and then the potential of the cell membrane in this piece of area is obtained by manual clamping, and the ion channel current on the membrane piece can be monitored and recorded.
3. The workflow of diaphragm clamp
(1) Prepare solution: Prepare the electrode inner and outer solution for cells and adjust the osmotic pressure and pH of the solution.
(2) Prepare samples: prepare cultured cells or brain slices.
(3) Draw glass microelectrodes: Draw glass capillaries and polish electrode tips with electrode drawing apparatus.
(4) Set up the perfusion system: Make sure the system is shielded.
(5) Clamp the cells: Use the 3D manipulator to control the electrodes to contact the cell membrane and ensure that the cells form a high resistance seal.
(6) Signal acquisition and amplification: To obtain the best experimental results, ensure that the correct type of amplifier is used for the study.
(7) Signal digital-to-analog conversion: Digital conversion of analog signals is performed in order to analyze the signals.
(8) Data acquisition and analysis: Using the pCLAMP 11 software, different programming and different experimental steps are designed according to the experimental purpose in order to perform faster data analysis and obtain more accurate determinations.
Figure 2 Working process of patch clamp
4. Basic Recording Modes of the Diaphragm Clamp Technique
There are four basic recording modes for the membrane clamp technique.
(1) Cell-attached recording mode;
When the glass electrode is moved by the microcontroller and its tip is lightly pressed against the cell membrane, a negative pressure is applied to the electrode to make the cell membrane sink into the glass electrode port, thus forming an "omega" seal with the inner wall of the electrode. At this time, the resistance between the electrode interior and the bath liquid can reach 1-100GΩ, which is a gi-ohm seal and can separate the adsorbed membrane from the bath liquid. After performing geo-sealing, the sealing can be firmly maintained even if the negative pressure inside the electrode is withdrawn.
(2) Inside-out recording mode;
An adherent recording configuration is formed first, and then the electrode is lifted on top of it. Due to the presence of a high-resistance seal, the contact between the open edge of the electrode tip and the cell membrane is quite strong, so that a piece of membrane will be torn off. In the low calcium solution, the side of this membrane piece originally facing the cytoplasm will be facing the bath solution.
(3) outside-out recording;
The electrode is lifted on the basis of the whole-cell recording configuration. The membrane around the tip of the glass electrode is torn off and the free edges of the cell membrane attached to the electrode are fused to each other. At this time, the original outer side of the cell membrane is still facing the bath solution.
(4) Whole-cell recording mode (whole-cell recording).
Based on the formation of the applanation recording configuration, the pulsating negative pressure is further applied inside the glass electrode, or (and) a single pulse click of appropriate amplitude and width is applied, so that the diaphragm sucked by the tip of the glass electrode is ruptured and the fluid inside the electrode and the cell is connected internally.
图3 膜片钳技术记录模式
5. Application of diaphragm clamp technique
Neher and Sakmann invented the membrane clamp technique in 1976, which can be used to detect the electrophysiological properties of cells. After more than 40 years of development, the membrane clamp technique has been widely used in the field of cell electrophysiology research.
(1) Studies of cell secretion;
(2) Studies of myocardial ion channels related to drug action;
(3) Studies on the physiological and pathological mechanisms of ion channel action;
(4) Studies on the relationship between single cell morphology and function;
(5) Studies on the mechanism of drug action;
(6) Studies in cardiovascular pharmacology;
(7) Studies of innovative drug and high throughput screening;
(8) Studies in the field of neuroscience.