This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.
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Method Article
Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes
In This Article
Summary
Abstract
Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.
Protocol
Part 1: Removing the vitelline membrane
- Prepare two sets of forceps. We prefer No. 5 forceps, where one set has been slightly sharpened with a file. Also prepare a glass transfer pipette by trimming and fire-polishing a 7" Pasteur pipette.
- Recommended: To prevent the oocytes from slipping, cut a circle of grid (1 mm squares) and glue into the bottom of a 60mm Petri dish. 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 ....
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Discussion
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.k.a., "peeling"), use newly pulled pipettes protected from dust, smooth pipettes, positive pressure applied when entering the bath sol.......
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Acknowledgements
Shrinking solution recipe is from the lab of R.W. Aldrich. 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. and Delia E. Baxter Foundation, the Klingenstein Fund and the McKnight Endowment for Neuroscience.
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Materials
| Name | Company | Catalog Number | Comments | |
|---|---|---|---|---|
| Patch Clamp Amplifier & Software | Instrument | HEKA Instruments | EPC-10 | Or other similar device (e.g. HEKA EPC-9 or Axopatch 200B) |
| No. 5 forceps (two pairs) | Tool | Fine Science Tools | ||
| Oocyte shrinking solution | Reagent | Ingredient In mMN-methyl-D-glucamine 220HEPES 10MgCl2 1EGTA 10Aspartic Acid 220KCl 2pH to 7.4 w/ N-methyl-D-glucamine | ||
| woven mesh | 800 um | Spectrum Labs | 146481 |
References
- Neher, E., Sakmann, B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature. 260, 799-802 (1976).
- Sackmann, B., Neher, E. Single-channel Recording. , Plenum Press. New York. (1983).
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