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Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor

We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after a...

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Autores principales: Wang, Xiaowei, Cato, Paul, Lin, Hsiu-Chen, Li, Tongen, Wan, Daniel, Alcocer, Marcos J. C., Falcone, Franco H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912355/
https://www.ncbi.nlm.nih.gov/pubmed/23893250
http://dx.doi.org/10.1007/s12033-013-9689-x
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author Wang, Xiaowei
Cato, Paul
Lin, Hsiu-Chen
Li, Tongen
Wan, Daniel
Alcocer, Marcos J. C.
Falcone, Franco H.
author_facet Wang, Xiaowei
Cato, Paul
Lin, Hsiu-Chen
Li, Tongen
Wan, Daniel
Alcocer, Marcos J. C.
Falcone, Franco H.
author_sort Wang, Xiaowei
collection PubMed
description We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after activation. In order to preserve cell binding, we tested the cell adhesion characteristics of different extracellular matrix proteins: human collagen I, fibronectin (FN) from bovine plasma and laminin (LN). FN was more effective than LN and collagen. Cell detachment after activation was in part due to reduced surface expression of VLA-4, the main ligand for FN, which was significantly decreased within 15 min of stimulation with 1 μg/mL calcium ionophore A23187, reaching a minimum after 2 h then slowly recovering. These optimised conditions were used for testing of well-characterised sera from allergic patients using two newly developed rat basophil leukaemia stable reporter cell lines (RBL NF-AT/GFP and RBL NF-AT/DsRed), which both express the human high-affinity IgE receptor alpha chain (FcεRIα). Both cell lines were able to detect sensitisation to specific allergens showing the expected bell-shaped dose–response curve, and correlated (R (2) = 0.75) with the standard beta-hexosaminidase assay, which is not suitable for an array format.
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spelling pubmed-39123552014-02-06 Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor Wang, Xiaowei Cato, Paul Lin, Hsiu-Chen Li, Tongen Wan, Daniel Alcocer, Marcos J. C. Falcone, Franco H. Mol Biotechnol Research We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after activation. In order to preserve cell binding, we tested the cell adhesion characteristics of different extracellular matrix proteins: human collagen I, fibronectin (FN) from bovine plasma and laminin (LN). FN was more effective than LN and collagen. Cell detachment after activation was in part due to reduced surface expression of VLA-4, the main ligand for FN, which was significantly decreased within 15 min of stimulation with 1 μg/mL calcium ionophore A23187, reaching a minimum after 2 h then slowly recovering. These optimised conditions were used for testing of well-characterised sera from allergic patients using two newly developed rat basophil leukaemia stable reporter cell lines (RBL NF-AT/GFP and RBL NF-AT/DsRed), which both express the human high-affinity IgE receptor alpha chain (FcεRIα). Both cell lines were able to detect sensitisation to specific allergens showing the expected bell-shaped dose–response curve, and correlated (R (2) = 0.75) with the standard beta-hexosaminidase assay, which is not suitable for an array format. Springer US 2013-07-27 2014 /pmc/articles/PMC3912355/ /pubmed/23893250 http://dx.doi.org/10.1007/s12033-013-9689-x Text en © The Author(s) 2013 https://creativecommons.org/licenses/by/2.0/ Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
spellingShingle Research
Wang, Xiaowei
Cato, Paul
Lin, Hsiu-Chen
Li, Tongen
Wan, Daniel
Alcocer, Marcos J. C.
Falcone, Franco H.
Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor
title Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor
title_full Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor
title_fullStr Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor
title_full_unstemmed Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor
title_short Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor
title_sort optimisation and use of humanised rbl nf-at-gfp and nf-at-dsred reporter cell lines suitable for high-throughput scale detection of allergic sensitisation in array format and identification of the ecm–integrin interaction as critical factor
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912355/
https://www.ncbi.nlm.nih.gov/pubmed/23893250
http://dx.doi.org/10.1007/s12033-013-9689-x
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