Cultured Cell Membrane Clamp Recording Experiment
Cultured Cell Membrane Clamp Recording Experiment
Source : Practical Laboratory Techniques in Neurobiology
Operation method
basic program
Principle
Cultured cell membrane-clamp recording is a method of separating tissue blocks into individual cells after mechanical separation and treatment with digestive enzymes under aseptic conditions and culturing them in an incubator for several days for routine membrane-clamp recording. The process of primary cell culture is basically the same as that of acute cell isolation, but the former is performed under sterile conditions.
Materials and Instruments
Cells Move 1. Cut the plastic coverslip into small slides with a side length of 5-8mm and spread them evenly in a 35mm plastic petri dish. 2. Treat the petri dish with 75% alcohol for 30 min, then wash it with sterilized double-distilled water for 1-2 times, dry it and prepare it for use. 3 Before culturing the cells, Id treat the Petri dishes with PDL (poly-D-lysine) and Laminin for 1 time each, and wash them with double-distilled water for 1 time in between. 4. All instruments used for extraction were sterilized in 75% alcohol for at least 30 min, an ice box was prepared, the digestive enzymes were removed and brought to room temperature, Puck's solution was pre-cooled, and F-12media was incubated in a 37'C water bath and set aside. 5. Animals were executed, and then the material was taken on the ice table. The extracted tissues were rinsed quickly to minimize erythrocyte contamination and then placed in cold Puck's solution. 6. Unwanted parts were removed with pre-sterilized fine scissors, and the tissue blocks were cut up and transferred to 15 ml centrifuge tubes. 7. Add lOng/ml papain or trypsin to digest at 37°C for 10-15min. 8. Centrifuge at low speed for 1min,discard the supernatant. 9 Add collagenase (1mg/ml) and dispase]](2.5mg/ml) and digest at 37℃ for 10-15min. 1O. Centrifuge at low speed for 1min, discard the supernatant. 11 Add 5 ml of pre-warmed F-12medium, mix well and centrifuge again for 1min, discard the supernatant. 12 Add F-l2media containing NGF (30ng/ml), gently blow 3-5 times with a pre-fired glass pipette to separate the cells evenly, and if necessary, use different caliber pipettes to blow in 2 times. 13. Transfer the separated cells to a 35-mm Petri dish with small slides, and then incubate them in an incubator for membrane clamp recording. Generally, the incubation time is not more than 1 week can be used for film clamp experiments, but need to change the culture medium every other day to ensure that the cells can get good nutrition and status. 14 Carefully place a small slide with cells attached to the wall into the small slot for membrane clamp recording and turn on the perfusate to perform routine membrane clamp experiments. The state of the cultured cells varies depending on the type of cells cultured and the type of digestive enzymes used, and the state of the cells used for membrane clamp recording is somewhat more demanding. Better cultured cells have a moderate density with less cellular debris and impurities (Figure 3-3). When performing membrane clamp experiments, we generally have to pay attention to the selection of healthy cells, which have a more regular morphology, a clean and smooth surface, a better adherence to the wall, and a three-dimensional sense under the microscope. Caveat l It is especially important to emphasize that all cell operations must be carried out on the ultra-clean bench, and all surgical instruments, liquids need to be sterilized or disinfected to strictly ensure that the cells are not contaminated.2 According to the experimental requirements to determine the age, sex, weight of the culture cell animals, but usually the younger the age of the animal, the stronger the cell tolerance, the better the activity. In addition, in order to ensure the activity of neurons, the fetching time should be as short as possible. 3. Adjust the type and whole of digestive enzymes according to the age of the animal. Fetal rats have fewer fibers in the nerve tissue, and only trypsin can be used for digestion; as young rats grow, the nerve fibers in the nerve tissue gradually increase, and need to be supplemented with collagenase digestion. There are also DNA enzymes and papain that can aid in digestion. Collagenase and DNA enzyme, papain digestion ability is weak, while trypsin digestion ability is strong, so the amount of trypsin can be less. Specific experiments should refer to the relevant literature and adjust the type and amount of digestive enzymes according to the different nerve tissues.4 In the process of mechanical separation of cells, the blowing of the tissue block must be light, because at this time, some cells have already been digested and separated, so in order to avoid this part of the cells from being damaged, it is best to use different caliber pipettes to blow in stages to ensure that more healthy and usable cells can be collected.5 Diaphragm clamp experimental process, especially for thousands of beginners, often encountered the main problem is the sealing difficulties, that is, can not form a giant resistance sealing, this time, you can check the cleanliness of the bath, such as turbidity, dust, impurities, etc., you need to replace the liquid or to keep the bath liquid flow. The glass microelectrode should be used within 2-3h after pulling and dust-proof, and the liquid inside the electrode must be filled after filtering. Check the tightness of the system composed of electrode holder, electrode and negative pressure suction tube, if there is no air leakage from the negative pressure suction tube, it means that the tightness is good, it is likely that the rubber pad in the electrode holder is not intact, and it should be replaced, and pay attention to the aperture of the rubber pad and the outer diameter of the electrode should be matched. If the cell surface is not smooth, it can also cause sealing difficulties, need to improve the degree of enzyme digestion in single-cell specimen preparation. The next problem encountered is that when the membrane is broken, the cells are easy to separate from the electrode, which indicates that the toughness and elasticity of the cell membrane is poor, and the cell state may be bad, and it is necessary to improve the method of specimen preparation to ensure the activity of the cells. The electrode can also be used after polishing. Another problem is that after the electrode is in the liquid, the current baseline of the sealing test is unstable and jittering violently, the perfusion system of the bath should be checked and adjusted to eliminate the vibration of the liquid level. Check the stability of the gripper to ensure that the electrode does not move when clamped. Check whether the silver wire part of the bath ground wire and the color of the silver wire in the gripper are whitish, and chlorinate them in time to ensure that the bath ground wire conducts electricity well. Common Problems 1 For cultured cells, the state of the cells is crucial, so it is very important for the selection and dosage of enzymes in the digestion process and the control of digestion time. If digestion is insufficient, the cell surface is rough or covered with adherents, and giant blocking sealing is difficult. If the digestion is excessive, the cell surface is clean and smooth, and the sealing is easy, but the elasticity of the cell membrane is poor, the cell is easy to be separated from the electrode after sealing and during the recording process, and the survival time of the cell is also short. Therefore, the digestion conditions must be adjusted for different specimens, and the appropriate amount of digestive enzymes and digestion time should be established after several experiments. Secondly, during the digestion process, the centrifuge tube can be gently shaken every 5 min, so that the tissue block and the enzyme can be fully mixed to achieve a better digestion effect. Finally, it is best to change the fluid for the cells 1d before recording to ensure that the cells have enough nutrition before recording to maintain the best condition. For more product details, please visit Aladdin Scientific website.
Cell culture medium is generally F-12medium Cell isolation process is generally Puck's solution Digestive enzymes trypsin or papain (10ng ml) Collagenase (1mg ml) Membrane Clamps
Diaphragm clamp amplifiers Micromanipulators Anti-vibration tables Microscopes Centrifuges Cell culture incubators, etc. Other accessories include 35mm cell culture dishes, centrifuge tubes, swimmer's forceps, etc.