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The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in. Automatic patch clamping. Automated patch clamp systems have recently been developed. Patch Clamp Electrophysiology. JoVE, Cambridge, MA, doi: 10.3791/5202 (2016). The patch clamp technique can be applied to study the function of ion channels that are activated by binding to signaling. Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes. This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes. Also prepare a glass transfer pipette by trimming and fire- polishing a 7. This dish can be reused indefinitely if kept clean between uses. Fill the Petri dish halfway with hyperosmotic solution (see recipe) and place under a dissecting microscope. Remove 1- 3 Xenopus oocytes from their incubation solution with a polished Pasteur pipette and place into the dish. Wait 1. 5s - 2 min for cells to begin to shrink. Longer times make cells easier to peel; shorter times lead to healthier cells for patching. As the cell shrinks, the vitelline membrane will begin to become barely visible as a transparent layer over the cell. Select a healthy cell (without whorls or defects) for peeling. With one pair of forceps, gently grasp the vitelline membrane without damaging the plasma membrane. With the other, grasp near the same spot and gently tear the clear membrane apart and free of the cell. With the Pasteur pipette, carefully move the cell to the recording chamber. Note that the cell will be fragile after vitelline removal. Part 2: Gigaseal generation. Patch-Clamp Analysis ADVANCED TECHNIQUES Second Edition Edited by Wolfgang Walz Department of Physiology, University of Saskatchewan. Fill a recording pipette with saline. Use the minimum volume of solution that makes good electrical contact with the electrode wire in order to minimize pipette capacitance. Flick the pipette several times to allow bubbles to float and slide onto the electrode wire. Apply pressure to the filled pipette. This can be done by mouth or with an aquarium pump from a pet store. This pressure is critical to keep the pipette cleans as it crosses the liquid interface and moves to the cell. Find the oocyte in the chamber and focus sharply on the edge of the cell.
With the pipette out of the bath, center the (out of focus) tip above the cell. This minimizes time spent in the bath before seal generation. Drop the pipette down into sharp focus next to the cell. Bring it into close proximity (~5. You should see a small distortion of solution pushed out of the pipette by the back pressure. Using the patch clamp software, check the resistance of the pipette (our goal is approximately 3- 4 M. As the tip begins to touch the cell, resistance will increase. Suddenly but gently switch from positive to negative pressure of approximately the same absolute value. Resistance should increase to seal formation. The majority of the time, seal formation occurs within several seconds; occasionally 3. Once the resistance is at 1. G. You now have an on- cell patch. For an inside- out patch, pull away from the cell rapidly. For outside- out, see below. Part 5: Outside- out topology (optional)Immediately after achieving a gigaseal, rupture the membrane. This can be done with sharp suction, but we prefer a 1 V pulse for 1 ms. The resistance should drop to slightly more than the initial pipette resistance. Move the pipette away from the cell slowly and smoothly. You should see a section of the cell pull with the pipette. Suddenly, and within a few seconds, the cell will snap back and the resistance should immediately and simultaneously return to G. The ectodomains of any included ion channels is exposed to the bath. Part 6: Anticipated Results. Generally, higher resistance seals are preferable and longer lived. In our experience, the chances of getting seals of this quality vary with many factors, particularly cell and pipette quality. Patch acquisition rates can be over 9. These patches may last for many minutes and are suitable for any electrophysiological study of ion channels expressed in Xenopus oocytes, including single- channel recordings. Subscription Required. Please recommend Jo. VE to your librarian. There are many parameters of electrophysiological recording not discussed here. Rig setup, system noise management, channel expression, and recording protocols are all also critical to good experimental results. In our experience, these are the most critical parameters for forming reliable seals: high quality oocytes, removing the vitelline membrane quickly (a. This being said, experience varies widely and others have their own sets of most important factors. Subscription Required. Please recommend Jo. VE to your librarian. Shrinking solution recipe is from the lab of R. W. We thank the following funding agencies and foundations for support: National Institutes of Health, National Science Foundation, American Heart Association, Muscular Dystrophy Association, the Donald B. Baxter Foundation, the Klingenstein Fund and the Mc. Knight Endowment for Neuroscience. Name. Type. Company. Catalog Number. Comments. Patch Clamp Amplifier & Software. Instrument. HEKA Instruments. EPC- 1. 0Or other similar device (e. HEKA EPC- 9 or Axopatch 2. B)No. 5 forceps (two pairs)Tool. Fine Science Tools. Oocyte shrinking solution. Reagent. Ingredient In m. MN- methyl- D- glucamine 2. HEPES 1. Mg. Cl. 2 1. EGTA 1. Aspartic Acid 2. KCl 2p. H to 7. 4 w/ N- methyl- D- glucaminewoven mesh. Spectrum Labs. 14. Neher, E., Sakmann, B. Single- channel currents recorded from membrane of denervated frog muscle fibres. Nature. 2. 60, 7. Sackmann, B., Neher, E. Single- channel Recording. My excitement about going to Japan turned into anxiousness when I heard that I was the only international student who got the opportunity to go on this lifetime study abroad adventure with 14 domestic students. Currently, Osaka University has concluded 102 inter-university and 492 inter-faculty academic exchange agreements (as of February 1, 2015). Students of our Program can apply for short- or long-term periods of study abroad. Spring Semester (January 25 to May 13, 2016)
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