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; Falcone, Franco H.

doi:10.1007/s12033-013-9689-x

Cited by 16 publications

(15 citation statements)

References 32 publications

(41 reference statements)

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“…More importantly, the RS‐ATL8 is not suitable for assessment of activation on allergen arrays, as luciferase detection requires cell lysis, thus disconnects the fluorescent signal from the location of the allergen spot causing activation on an array slide. To overcome this limitation, we developed a fluorescent reporter system, which similarly to the RS‐ATL8, reports IgE‐dependent NFAT nuclear translocation by inducing DsRed protein expression (NFAT‐DsRed RBL) …”

Section: The New Generation Of Rbl Cell‐based Modelsmentioning

confidence: 99%

RBL cells as models for in vitro studies of mast cells and basophils

Falcone

Wan

Barwary

et al. 2018

Immunological Reviews

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Since their establishment in 1981, RBL-2H3 cells have been widely used as a mast cell (MC) model. Their ability to be easily grown in culture in large amounts, their responsiveness to FcεRI-mediated triggers and the fact that they can be genetically manipulated, have provided advantages over primary MCs, in particular for molecular studies relying on genetic screening. Furthermore, the ability to generate clones that stably express proteins of interest, for example, a human receptor, have marked the RBL cells as an attractive MC model for drug screening. Indeed, 3 RBL reporter cell lines (RS-ATL8, NFAT-DsRed, and NPY-mRFP) have been generated providing useful models for drug and allergen screening. Similarly, RBL cells stably expressing the human MrgprX2 receptor provide a unique paradigm for analyzing ligand interactions and signaling pathways of the unique human receptor. Finally, transient co-transfections of RBL cells allow functional genomic analyses of MC secretion by combining library screening with simultaneous expression of a reporter for exocytosis. RBL cells thus comprise powerful tools for the study of intracellular membrane trafficking and exocytosis and the detection of allergens, vaccine safety studies and diagnosis of allergic sensitization. Their recent uses as an investigative tool are reviewed here.

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Section: The New Generation Of Rbl Cell‐based Modelsmentioning

confidence: 99%

RBL cells as models for in vitro studies of mast cells and basophils

Falcone

Wan

Barwary

et al. 2018

Immunological Reviews

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“…The most recent generation of transgenic cell lines has further modified these humanized RBL lines to include sensitive and easy to measure reporter genes, such as firefly luciferase (RS-ATL8; [11]) or red fluorescent protein (NFAT-DsRed; [12]). These reporter cell lines have a series of advantages over the older generation, which we have described in detail in a recent review [3].…”

Section: Introductionmentioning

confidence: 99%

Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines

et al. 2019

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Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRI H ). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRI H surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay–a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRI H chain in humanized RBL cell lines. We compared surface levels of FcεRIα H by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIα H copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγ H cDNA was assessed for its ability to increase FcεRIα H expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703–21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIα H expression, respectively. This was neither related to FcεRI H gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIα H surface expression appeared to correlate with the co-expression of FcεRIγ H . Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγ H , increased FcεRIα H chain expression levels. Levels of FcεRIα H surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

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“…Experiments performed with cell penetrating luciferase substrate formulations were attempted, but not further pursued. As a viable alternative, we developed a reporter system expressing intracellular fluorescence (RBL 703/21 NFAT-DsRed) showing high stability in solid-phase assays 16 . In order to establish proof-of-principle that our fluorescent reporter cell line could be used in printed array format, we first needed to investigate several parameters regarding printed protein stability before and after washing the surfaces, cell binding to solid surfaces, and the ability of different polymer coatings to support cell activation.…”

Section: Discussionmentioning

confidence: 99%

“…Cell culture. All experiments were performed with rat basophilic leukaemia cells (RBL-703/21-derived NFATp-DsRed-Express2) generated at The University of Nottingham 16 . These are available from the authors for non-commercial purposes on the basis of a material transfer agreement (MTA).…”

Section: Methodsmentioning

confidence: 99%

“…However, purifying basophils from peripheral blood of human donors is not a widely acceptable proposition for non-specialised labs, despite strongly improved and semi-automated protocols for their purification 15 . We and others have previously suggested replacing human basophils with humanised rat basophilic leukaemia (RBL) reporter cell lines 16 – 19 . RBL cells were first introduced in 1981 and have been used for many decades as a mast cell model to investigate the molecular basis of IgE-dependent signal transduction 20 .…”

Section: Introductionmentioning

confidence: 99%

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Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis

Kalli

Blok

Jiang

et al. 2020

Sci Rep

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Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF–SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

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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

Cited by 16 publications

References 32 publications

RBL cells as models for in vitro studies of mast cells and basophils

RBL cells as models for in vitro studies of mast cells and basophils

Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis

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