Abstract
RationaleUnbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.ObjectivesValidation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.MethodsWe analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.Measurements and Main ResultsWe observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.ConclusionCFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.
Highlights
Quantitative protein analysis at single cell level is critically important to study cell-type specific regulation of protein function in health and disease but limited techniques are available to perform single cell analysis in primary patient material [1,2]
CFTR protein is expressed in Cystic fibrosis (CF) patients harboring F508del mutations but at lower levels than in healthy controls
Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level
Summary
Quantitative protein analysis at single cell level is critically important to study cell-type specific regulation of protein function in health and disease but limited techniques are available to perform single cell analysis in primary patient material [1,2]. Contrasting data has been published on F508del CFTR protein expression levels in native airway epithelial cells. Kalin et al showed endogenous wild type (wt) and F508del CFTR at similar intensity levels as healthy controls at the apical membrane in epithelial from nasal polyps [9]. This is in accordance with a study published by Penque et al who observed apical CFTR in nasal epithelial cells from homozygous F508del patients, it was found that the percentage of CFTR positive cells were significantly lower [10]. Quantification of CFTR protein expression has been proven difficult and it remains unclear whether differences in CFTR expression levels of individual patients can be related to residual function and CF disease variability in subjects harboring similar CFTR mutations
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