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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| Format: | Article |
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Humana Press
2014
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| Online Access: | https://eprints.nottingham.ac.uk/2916/ |
| _version_ | 1848790908308291584 |
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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 |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| 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. |
| first_indexed | 2025-11-14T18:20:05Z |
| format | Article |
| id | nottingham-2916 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T18:20:05Z |
| publishDate | 2014 |
| publisher | Humana Press |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-29162020-05-04T20:15:38Z https://eprints.nottingham.ac.uk/2916/ 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. 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. Humana Press 2014-02 Article PeerReviewed Wang, Xiaowei, Cato, Paul, Lin, Hsiu-Chen, Li, Tongen, Wan, Daniel, Alcocer, Marcos J.C. and Falcone, Franco H. (2014) 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. Molecular Biotechnology, 56 (2). pp. 136-146. ISSN 1073-6085 RBL Basophil array Adhesion Fibronectin VLA-4 Reporter system GFP DsRed Intro http://link.springer.com/article/10.1007%2Fs12033-013-9689-x doi:10.1007/s12033-013-9689-x doi:10.1007/s12033-013-9689-x |
| spellingShingle | RBL Basophil array Adhesion Fibronectin VLA-4 Reporter system GFP DsRed Intro 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 | RBL Basophil array Adhesion Fibronectin VLA-4 Reporter system GFP DsRed Intro |
| url | https://eprints.nottingham.ac.uk/2916/ https://eprints.nottingham.ac.uk/2916/ https://eprints.nottingham.ac.uk/2916/ |