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Development of a high-throughput fluorescent no-wash sodium influx assay
Voltage-gated sodium channels (Na(V)s) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dy...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411159/ https://www.ncbi.nlm.nih.gov/pubmed/30856233 http://dx.doi.org/10.1371/journal.pone.0213751 |
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author | Tay, Bryan Stewart, Teneale A. Davis, Felicity M. Deuis, Jennifer R. Vetter, Irina |
author_facet | Tay, Bryan Stewart, Teneale A. Davis, Felicity M. Deuis, Jennifer R. Vetter, Irina |
author_sort | Tay, Bryan |
collection | PubMed |
description | Voltage-gated sodium channels (Na(V)s) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495–575 nm) to assays in HEK293 cells heterologously expressing hNa(V)1.1–1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on Na(V) channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the Na(V)1.1–1.7 activator veratridine (EC(50) 10–29 μM) or the Na(V)1.1–1.8 inhibitor tetracaine (IC(50)’s 6–66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R. |
format | Online Article Text |
id | pubmed-6411159 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-64111592019-04-01 Development of a high-throughput fluorescent no-wash sodium influx assay Tay, Bryan Stewart, Teneale A. Davis, Felicity M. Deuis, Jennifer R. Vetter, Irina PLoS One Research Article Voltage-gated sodium channels (Na(V)s) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495–575 nm) to assays in HEK293 cells heterologously expressing hNa(V)1.1–1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on Na(V) channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the Na(V)1.1–1.7 activator veratridine (EC(50) 10–29 μM) or the Na(V)1.1–1.8 inhibitor tetracaine (IC(50)’s 6–66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R. Public Library of Science 2019-03-11 /pmc/articles/PMC6411159/ /pubmed/30856233 http://dx.doi.org/10.1371/journal.pone.0213751 Text en © 2019 Tay et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Tay, Bryan Stewart, Teneale A. Davis, Felicity M. Deuis, Jennifer R. Vetter, Irina Development of a high-throughput fluorescent no-wash sodium influx assay |
title | Development of a high-throughput fluorescent no-wash sodium influx assay |
title_full | Development of a high-throughput fluorescent no-wash sodium influx assay |
title_fullStr | Development of a high-throughput fluorescent no-wash sodium influx assay |
title_full_unstemmed | Development of a high-throughput fluorescent no-wash sodium influx assay |
title_short | Development of a high-throughput fluorescent no-wash sodium influx assay |
title_sort | development of a high-throughput fluorescent no-wash sodium influx assay |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411159/ https://www.ncbi.nlm.nih.gov/pubmed/30856233 http://dx.doi.org/10.1371/journal.pone.0213751 |
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