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A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427
BACKGROUND: Human African Trypanosomiasis (HAT) is caused by two trypanosome species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness acros...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2009
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781010/ https://www.ncbi.nlm.nih.gov/pubmed/19909542 http://dx.doi.org/10.1186/1756-3305-2-54 |
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author | Sykes, Melissa L Avery, Vicky M |
author_facet | Sykes, Melissa L Avery, Vicky M |
author_sort | Sykes, Melissa L |
collection | PubMed |
description | BACKGROUND: Human African Trypanosomiasis (HAT) is caused by two trypanosome species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness across species and disease stages, thus there is a considerable need to find alternative drugs. A well recognised approach to identify new drug candidates is high throughput screening (HTS) of large compound library collections. RESULTS: We describe here the development of a luciferase based viability assay in 384-well plate format suitable for HTS of T.b.brucei. The parameters that were explored to determine the final HTS assay conditions are described in detail and include DMSO tolerability, Z', diluents and cell inoculum density. Reference compound activities were determined for diminazene, staurosporine and pentamidine and compared to previously published IC(50 )data obtained. The assay has a comparable sensitivity to reference drugs and is more cost effective than the 96-well format currently reported for T.b.brucei. CONCLUSION: Due to the reproducibility and sensitivity of this assay it is recommended for potential HTS application. As it is commercially available this assay can also be utilised in many laboratories for both large and small scale screening. |
format | Text |
id | pubmed-2781010 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-27810102009-11-24 A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427 Sykes, Melissa L Avery, Vicky M Parasit Vectors Research BACKGROUND: Human African Trypanosomiasis (HAT) is caused by two trypanosome species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness across species and disease stages, thus there is a considerable need to find alternative drugs. A well recognised approach to identify new drug candidates is high throughput screening (HTS) of large compound library collections. RESULTS: We describe here the development of a luciferase based viability assay in 384-well plate format suitable for HTS of T.b.brucei. The parameters that were explored to determine the final HTS assay conditions are described in detail and include DMSO tolerability, Z', diluents and cell inoculum density. Reference compound activities were determined for diminazene, staurosporine and pentamidine and compared to previously published IC(50 )data obtained. The assay has a comparable sensitivity to reference drugs and is more cost effective than the 96-well format currently reported for T.b.brucei. CONCLUSION: Due to the reproducibility and sensitivity of this assay it is recommended for potential HTS application. As it is commercially available this assay can also be utilised in many laboratories for both large and small scale screening. BioMed Central 2009-11-12 /pmc/articles/PMC2781010/ /pubmed/19909542 http://dx.doi.org/10.1186/1756-3305-2-54 Text en Copyright ©2009 Sykes and Avery; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Sykes, Melissa L Avery, Vicky M A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427 |
title | A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427 |
title_full | A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427 |
title_fullStr | A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427 |
title_full_unstemmed | A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427 |
title_short | A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427 |
title_sort | luciferase based viability assay for atp detection in 384-well format for high throughput whole cell screening of trypanosoma brucei brucei bloodstream form strain 427 |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781010/ https://www.ncbi.nlm.nih.gov/pubmed/19909542 http://dx.doi.org/10.1186/1756-3305-2-54 |
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