A recent paper demonstrates how peripheral blood mononuclear cells (PBMCs) can be used to detect CFTR protein in an easy and non-invasive way by flow cytometry.
Cystis fibrosis transmembrane conductance regulator (CFTR) gene encodes for an anion channel in the epithelial cells. Dysfunction of CFTR protein due to mutations in the gene results in cystic fibrosis, a debilitating disease. CFTR is also expressed in several types of cells in PBMCs, including lymphocytes and macrophages and its main function in these cells is regulation of membrane potential. Dysfunction of CFTR in PBMCs specifically affects chloride ion flow and the ability of these cells to engage in inflammation and other bactericidal activities.
CFTR is a membrane protein and dysfunction can be due to: a. lack of protein synthesis, b. improper trafficking (leading to mis-localization) or c. improper channel function, in spite of being at the membrane. CFTR mutations are classified into five different mutations, which affect one of the three above categories. Just as with any other disease, there are continual efforts for easy detection of cystic fibrosis. Among the several approaches, drawing of peripheral blood for diagnosis is the one of the least invasive methods available. Currently, there is one such method for detection of cystic fibrosis, caused by class I mutation which results in impaired protein synthesis in PBMCs. The current protocol requires isolation of high number of PBMCs, followed by Western blot analysis, which is only semi-quantitative. A recent paper by Johansson et al., describes a flow cytometry protocol, which requires relatively less amounts of PBMCs and is also quantitative.In this paper, the authors:
1. Identified the membrane presence of CFTR in PBMCs, also available from HemaCare, by Western blot.
2. Used PBMCs from both healthy controls and cystic fibrosis patients to look for sub-cellular localization. They found co-localization of CFTR with CD14 (a membrane protein) in controls, but not in patient samples (Fig 1.).
3. Employed flow cytometry to distinguish PBMCs in which CFTR and CD14 are co-localized, i.e. from healthy and diseased samples. From a series of experiments, they assigned mean fluorescence intensity (M.F.I) to monocytes in PBMCs.
Thus, they have developed a fairly easy protocol to diagnose cystic fibrosis using a few hundred microlitres of blood. The success of the protocol is ensured both by high sensitivity and reproducibility. Further, the authors demonstrated how the protocol can also be used to test the efficacy of cystic fibrosis treatment drugs, e.g. ataluren, as flow cytometry experiments performed with patient samples, before and after ataluren treatment showed significant differences. For obtaining whole blood or PBMCs to suit you specific research needs, turn to HemaCare, a leading provider of blood products from both healthy and patient donors!
1. Jan Johansson, Marzia Vezzalini, Genny Verze, Sara Caldrer, Silvia Bolognin, Mario Buffelli, Giuseppe Bellisola, Gloria Tridello, Baroukh Maurice Assael, Paola Melotti, Claudio Sorio. Detection of CFTR Protein in Human Leukocytes by Flow Cytometry. Cytometry. 2014 doi: 10.1002/cyto.a.22456