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Published on: June 22, 2022
A novel fluorescent sensor for measurement of CFTR function by flow cytometry
Lodewijk A W Vijftigschild1, Cornelis K van der Ent, Jeffrey M Beekman
1Department of Pediatric Pulmonology, Wilhelmina Children's Hospital, University Medical Centre Utrecht, Utrecht, The Netherlands.
Summary
This study introduces a new flow cytometry method to accurately measure cystic fibrosis transmembrane conductance regulator (CFTR) function. Ratiometric measurements using co-expressed fluorescent proteins enable precise CFTR activity quantification in suspension cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
- Studying CFTR function typically involves measuring iodide transport using halide-sensitive yellow fluorescent protein (YFP), but this is sensitive to expression levels.
- Existing methods require normalization of YFP expression, which can be challenging for accurate quantification.
Purpose of the Study:
- To develop a flow cytometry-based assay for accurate measurement of CFTR function.
- To establish a ratiometric method for normalizing sensor expression levels in CFTR function assays.
- To enable the study of CFTR function in suspension cells and identify CFTR mutants with differential residual function.
Main Methods:
- Co-expression of halide-sensitive YFP with iodide-insensitive fluorescent proteins (dsRed, mKate) in cells expressing wild-type or mutant CFTR.
- Development of a ratiometric measurement (YFP/mKate) to correct for variations in sensor expression.
- Application of ratiometric YFP quenching for CFTR function analysis by flow cytometry.
Main Results:
- dsRed and mKate fluorescence were confirmed to be iodide-insensitive.
- Ratiometric measurements using YFP/mKate effectively normalized for differences in sensor expression levels.
- The flow cytometry assay could distinguish as little as 10% of wild-type CFTR expressing cells.
- The method accurately quantified CFTR function for mutants with differential residual function.
Conclusions:
- Ratiometric measurement by flow cytometry provides an accurate method for quantifying CFTR function.
- This novel approach allows CFTR function studies in suspension cells, overcoming limitations of plate-bound assays.
- The technique can be adapted for cell sorting and studying CFTR in complex cellular environments.
