Background: Simplified methods for CFTR detection suitable for the evaluation of response to CFTR-targeting drugs are requested to evaluate drug response. Methods: We applied flow cytometry (FC) analysis to detect CFTR expression in CFBE41o-, a bronchial epithelial cell line homozygous for F508del mutation, and 16HBE14o-, from healthy donor, that are used to test molecules targeting CFTR defect. As additional parameter we also evaluat- ed CFTR activity by single-cell fluorescence assay. CFBE41o- cells were treated with VRT325 (10μM), VX-809 (5μM) or the drugs’ vehicle DMSO for 24h. FC analysis was performed with the anti-CFTR monoclonal anti- body CF3 (Abcam 2784, specific for the extracellular domain of CFTR: aa 103-117) and with the anti-CFTR polyclonal antibody ACL-006 (Alomone labs, specific for the intracellular domain of CFTR: aa 1468-1480). We selected two parameters to quantify CFTR expression: Mean fluorescence intensity (MFI) ratio value and % CFTR-positive cells. CFTR activity was assayed with the potential-sensitive probe (DiSBAC2(3), Invitrogen, USA). We defined “CF index” as a parameter that is positive in non-CF cells and negative in CF cells (Sorio, et al. PLoS One. 2011;6:e22212). Results: A higher percentage of CFTR-positive cells was recorded in CFBE41o- cells after 24h treatment with VRT325 with respect to non- treated cells while VX-809 apparently is not as effective as VRT325 in these experimental conditions. These results were related to the measure of CFTR function recorded with cell depolarization assay. We already pub- lished a similar setup utilized for leukocytes (Johansson J, et al. Cytometry A. 2014;85:611-20) with antibody ACL-006 and now we are testing the performance of CF3 antibody in leukocytes. Conclusion: Optimization of the protocol is in progress. Nevertheless the method described is proved capable to detect correction of CF pheno- type, is simple and rapid. It may be applied to primary cells to monitor the responses to drugs whose efficacy can depend on increased CFTR protein expression or processing to the cell surface. Supported by: FFC #26-2011, #06-2013, #29-2015, Associazione Lega Italiana FC.

Poster Session Abstracts

Vercellone, Silvia;Caldrer, Sara;BUFFELLI, Mario Rosario;MELOTTI, Paola Maria;SORIO, Claudio
2016-01-01

Abstract

Background: Simplified methods for CFTR detection suitable for the evaluation of response to CFTR-targeting drugs are requested to evaluate drug response. Methods: We applied flow cytometry (FC) analysis to detect CFTR expression in CFBE41o-, a bronchial epithelial cell line homozygous for F508del mutation, and 16HBE14o-, from healthy donor, that are used to test molecules targeting CFTR defect. As additional parameter we also evaluat- ed CFTR activity by single-cell fluorescence assay. CFBE41o- cells were treated with VRT325 (10μM), VX-809 (5μM) or the drugs’ vehicle DMSO for 24h. FC analysis was performed with the anti-CFTR monoclonal anti- body CF3 (Abcam 2784, specific for the extracellular domain of CFTR: aa 103-117) and with the anti-CFTR polyclonal antibody ACL-006 (Alomone labs, specific for the intracellular domain of CFTR: aa 1468-1480). We selected two parameters to quantify CFTR expression: Mean fluorescence intensity (MFI) ratio value and % CFTR-positive cells. CFTR activity was assayed with the potential-sensitive probe (DiSBAC2(3), Invitrogen, USA). We defined “CF index” as a parameter that is positive in non-CF cells and negative in CF cells (Sorio, et al. PLoS One. 2011;6:e22212). Results: A higher percentage of CFTR-positive cells was recorded in CFBE41o- cells after 24h treatment with VRT325 with respect to non- treated cells while VX-809 apparently is not as effective as VRT325 in these experimental conditions. These results were related to the measure of CFTR function recorded with cell depolarization assay. We already pub- lished a similar setup utilized for leukocytes (Johansson J, et al. Cytometry A. 2014;85:611-20) with antibody ACL-006 and now we are testing the performance of CF3 antibody in leukocytes. Conclusion: Optimization of the protocol is in progress. Nevertheless the method described is proved capable to detect correction of CF pheno- type, is simple and rapid. It may be applied to primary cells to monitor the responses to drugs whose efficacy can depend on increased CFTR protein expression or processing to the cell surface. Supported by: FFC #26-2011, #06-2013, #29-2015, Associazione Lega Italiana FC.
FLOW CYTOMETRY, CFTR, epithelium, leukocytes
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/955496
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