Avoiding the formation of vesicles by patch excision from Xenopus oocytes

作者：Thon S; Be***orf K^*

来源：Journal of Neuroscience Methods, 2014, 225: 29-31.

DOI：10.1016/j.jneumeth.2014.01.005

• Xenopus
• Inside-out patch
• Vesicle

Background: The patch-clamp technique is well-established to investigate the function of ion channels. Several patch configurations have been described, including the inside-out patch configuration providing the unique advantage of having free access to the patch from the cytosolic side. An inside-out patch is predominantly built from a cell-attached patch by pulling the patch pipette back. However, when using pipettes with high resistance (%26gt;8 M Omega), often a vesicle is formed instead of the desired inside-out patch, preventing proper recording of ion currents. %26lt;br%26gt;New method: Using quartz pipettes with high resistance we studied the benefit of a simple alternative excision manoeuvre that significantly enhances the efficiency to obtain an inside-out patch from Xenopus oocytes. %26lt;br%26gt;Results: We show that the formation of vesicles depends on the direction of patch excision: after a cell attached patch has formed, pushing the patch pipette first into the depth of the oocyte and exposing the patch only then to the bath solution generated a success rate of 89% (16 out of 18) for a proper inside-out patch, as evaluated by the current flowing through HCN2 channels which were heterologously expressed in the oocytes. %26lt;br%26gt;Comparison with existing method: In contrast, with the same type of pipettes and oocytes only 22% (4 out of 18) of the patches developed HCN2 currents when pulling the pipette in the backward direction as usual. %26lt;br%26gt;Conclusion: The difference in the success rate favours the idea to use %26quot;pushed inside-out patches%26quot; instead of %26quot;pulled inside-out patches%26quot; when studying ion channels expressed in Xenopus oocytes.

• 出版日期2014-3-30