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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

Wang, Xiaowei; Cato, Paul; Lin, Hsiu-Chen; Li, Tongen; Wan, Daniel; Alcocer, Marcos J.C.; Falcone, Franco H.

Authors

Xiaowei Wang

Paul Cato

Hsiu-Chen Lin

Tongen Li

Daniel Wan

Marcos J.C. Alcocer

Franco H. Falcone



Abstract

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.

Journal Article Type Article
Publication Date 2014-02
Journal Molecular Biotechnology
Print ISSN 1073-6085
Electronic ISSN 1559-0305
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 56
Issue 2
APA6 Citation Wang, X., Cato, P., Lin, H., Li, T., Wan, D., Alcocer, M. J., & Falcone, F. 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), https://doi.org/10.1007/s12033-013-9689-x
DOI https://doi.org/10.1007/s12033-013-9689-x
Keywords RBL, Basophil array, Adhesion, Fibronectin, VLA-4, Reporter system, GFP, DsRedIntro
Publisher URL http://link.springer.com/article/10.1007%2Fs12033-013-9689-x
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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