A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perf...

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Autores principales:	Chon Lok Lei, Alan Fabbri, Dominic G. Whittaker, Michael Clerx, Monique J. Windley, Adam P. Hill, Gary R. Mirams, Teun P. de Boer
Formato:	article
Lenguaje:	EN
Publicado:	Wellcome 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a53e5521451443e3b5ff249890662cc0
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spelling	oai:doaj.org-article:a53e5521451443e3b5ff249890662cc02021-11-15T15:27:33ZA nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]2398-502X10.12688/wellcomeopenres.15968.2https://doaj.org/article/a53e5521451443e3b5ff249890662cc02021-11-01T00:00:00Zhttps://wellcomeopenresearch.org/articles/5-152/v2https://doaj.org/toc/2398-502XAutomated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F− containing internal solution together with a Ca2+ containing external solution — so that a CaF2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F− ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.Chon Lok LeiAlan FabbriDominic G. WhittakerMichael ClerxMonique J. WindleyAdam P. HillGary R. MiramsTeun P. de BoerWellcomearticleMedicineRScienceQENWellcome Open Research, Vol 5 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Chon Lok Lei Alan Fabbri Dominic G. Whittaker Michael Clerx Monique J. Windley Adam P. Hill Gary R. Mirams Teun P. de Boer A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]
description	Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F− containing internal solution together with a Ca2+ containing external solution — so that a CaF2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F− ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.
format	article
author	Chon Lok Lei Alan Fabbri Dominic G. Whittaker Michael Clerx Monique J. Windley Adam P. Hill Gary R. Mirams Teun P. de Boer
author_facet	Chon Lok Lei Alan Fabbri Dominic G. Whittaker Michael Clerx Monique J. Windley Adam P. Hill Gary R. Mirams Teun P. de Boer
author_sort	Chon Lok Lei
title	A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]
title_short	A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]
title_full	A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]
title_fullStr	A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]
title_full_unstemmed	A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]
title_sort	nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]
publisher	Wellcome
publishDate	2021
url	https://doaj.org/article/a53e5521451443e3b5ff249890662cc0
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A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 2; peer review: 1 approved, 3 approved with reservations]

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