Data represent average SEM. == Discussion == Here, we describe a novel procedure to study CFTR expression in small amounts of primary human nasal epithelial cells using flow cytometry. 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 Results == We 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. == Conclusion == CFTR 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. == Introduction == 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]. Flow cytometry has been widely used in immunology to study protein expression at single cell level of haematopoietic cells. The application of flow cytometry for other tissues is hampered by the ability to generate single cell suspensions, and the accessibility of patient samples. Cystic fibrosis (CF) is caused by mutations of the gene encoding for Cystic Fibrosis transmembrane conductance regulator (CFTR)[3][5]. CF affects multiple organs but morbidity and mortality is dominated by CF lung disease that is characterized by mucus plugging, airway infections and sustained inflammation[6]. The most common mutation encodes for a CFTR protein that lacks phenylalanine at position 508 (F508del CFTR) causing it to misfold and retain in the endoplasmic reticulum from where is Triciribine phosphate (NSC-280594) degraded[7],[8]. Contrasting data has been published on F508del CFTR protein expression levels in native airway epithelial cells. Klinet 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 Penqueet al. who observed apical CFTR in nasal epithelial cells from homozygous F508del patients, although it was found that the percentage of CFTR positive cells were significantly lower[10]. In addition Borthwicket al.recently reported similar CFTR expression at the apical surface between non-CF and CF cells in bronchial epithelium, although in CF cells the amount of CFTR expression was reduced[11]. However, Kredaet al.could not detect F508del CFTR at the apical membrane, and reported that the immature form of CFTR that resides in the ER was present at much lower levels[12],[13]. So quantification of CFTR protein expression has been proven difficult and it remains unclear whether differences in Triciribine phosphate (NSC-280594) CFTR expression levels of individual patients can be related to residual function and CF disease variability in subjects harboring similar CFTR mutations. Here, we developed a novel unbiased procedure to study CFTR expression in primary epithelial cells isolated from the nasal cavity at the single cell level by flow cytometry. CFTR function measurements in nasal epithelium correlate with CF disease indicating that nasal epithelium is a relevant tissue for studying CF disease mechanisms[14]. Nasal epithelial cells were harvested by a relatively noninvasive simple procedure. We validated this technique using other previously described techniques including Western blot analysis and RT-PCR. With this procedure we were able to study CFTR expression in nasal epithelial cells with a variety of CFTR antibodies, and found F508del homozygous patients to express CFTR protein, but at a lower level compared to healthy controls. == Results == == Validation of CFTR expression in primary human epithelial cells == Since CFTR expression analysis by Western blot in cells Rabbit polyclonal to PKC alpha.PKC alpha is an AGC kinase of the PKC family.A classical PKC downstream of many mitogenic and receptors.Classical PKCs are calcium-dependent enzymes that are activated by phosphatidylserine, diacylglycerol and phorbol esters. obtained from the nasal cavity by brushing is difficult due to contaminating cells and low cell yield we developed a novel assay to study CFTR protein level in individual nasal epithelial cells. However, we first validated that cells isolated by Triciribine phosphate (NSC-280594) nasal brushing expressed CFTR as has been shown by others[10],[15],[16]. As expected, we observed CFTR mRNA expression in nasal cells obtained from two healthy individuals as indicated by RT-PCR (Fig. 1A). Calu-3 cells served as positive control and 2M was amplified as control for cDNA input. To assess CFTR protein expression in cells obtained from the nasal cavity, we pooled samples of multiple healthy individuals and performed Western blot analysis. We observed immunoreactivity around 170 kDa and to a lesser extent around 150 kDa in nasal cells and Calu-3 cells that co-migrated with ectopic CFTR expressed by BHK cells (Fig. 1B). This suggests that nasal cells collected by brushing expressed the fully glycosylated mature form (band C) and the immature form.