Walter Gassmann and Julian Schroeder
Department of Biology, University of California, San Diego
Schroeder Lab Protocol 1992, 1993
Nystatin Slow-Whole-Cell - On Wheat Root Hair Cells
1) Weigh out approximately 2.5 mg Nystatin (very light-sensitive) and dissolve in 100% DMSO to obtain a 25 mg/ml stock. Dissolve by vortexing for approx. 30 sec. Wrap stock in aluminum foil.
2) Just before start of the experiment, add nystatin to pipette solution, 200 ug/ml, i.e. 8 ul stock per ml of pipette solution. Remember to set aside some nystatin-free solution for tip-filling; getting a seal was not possible if nystatin was present at the tip. Wrap pipette solution in tin foil.
3) Fill tip of pipette to about 400 um with nystatin-free solution by dipping tip into the solution for approx. 1.5 sec. You can verify speed of capillary action of your particular pipette tip under a microscope with the pipette in air (i.e. fire-polishing scope), but do this with the actual pipette solution. The speed of filling by capillary varies with ionic content.
4) Backload the pipette with nystatin-containing pipette solution and get a seal as quickly as possible (e.g. within approx. 10 min. after backfilling nystatin). It appeared OK to apply suction to the pipette while making a seal. If you got a seal, after 10-30 min. after backfilling your pipette you should start to see changes in the capacitance signifying access to the cytosol. The access resistance decreases very slowly over time, as expected for gradual formation of a perforated patch. Cell capacitances were similar to those recorded with the conventional whole-cell patch clamp technique and whole-cell currents were recorded.
5) Keep the nystatin-pipette solution only for 2-4 h!; make the nystatin-DMSO stock fresh every day!
In our experiments this technique worked well several times in root hair cells from wheat. However, we were not successful at using it on carrot cell suspension protoplasts, in which we were studying voltage-dependent Ca2+ channels. It is possible that the membranes of different cells are sufficiently different to significantly affect the efficacy of nystatin function/incorporation. (One idea, that we did not test, is to determine the efficacy of nystatin in perforating a given cell line, by adding nystatin to the bath solution and following cell death because, one might expect perforation of entire cells to induce cell death).
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