1). In comparison, protein bands were observed at ∼150 kDa for all Calu-3 cell lysates and were the strongest

for cells at a high passage number cultured at the ALI (Fig. 1). The mouse anti-human MDR1 antibodies UIC2 and MRK16 were subsequently used for immunohistochemistry and flow cytometry. A positive immunohistochemical signal was obtained with both antibodies on the apical membranes of all but HEK293 cell layers investigated (Fig. 2). This was however discontinuous on NHBE and low passage Calu-3 layers (Fig. 2). Both MDCKII-WT and MDCKII-MDR1 cell layers stained positively, possibly due to the cross-reactivity of the antibodies with the canine mdr1 expressed in the cells [29]. Staining ABT-263 mouse GSK3 inhibitor appeared nevertheless more intense for the transfected cells. Flow cytometry using the UIC2 antibody produced a low MFI value of 1.3 with the negative control MDCKII-WT cells, whereas the MDCKII-MDR1 positive cell control generated a MFI value of 7.5, demonstrating the UIC2 antibody reacts specifically with MDR1. At low passage, 36% of Calu-3 cells were shown to express the MDR1 transporter in comparison with 70% at a high passage, resulting in a MFI of 5.2 and 15.0, respectively (Fig. 3). In contrast, only 6% of NHBE cells expressed MDR1 (MFI = 1.3). Similar trends in MDR1 expression levels were

obtained with the MRK16 antibody with, however, lower fluorescence values recorded, likely due to a weaker affinity of this antibody for MDR1 (Fig. S1; Supplementary information). The well-established MDR1 substrate digoxin is often used to probe MDR1 in biological systems, both in vitro and in vivo [13] and [17]. However, the drug has also been reported to be a substrate for other transporters detected at the gene level in our broncho-epithelial cell layers (e.g. some of the OATP) [20] and [21]. Hence, in order to verify the functionality of MDR1 in bronchial

epithelial cells, we performed an UIC2 antibody shift assay in presence of the much potent MDR1 inhibitor PSC833 as an alternative to measuring digoxin efflux ratios. This assay is based on the observation that binding of MDR1 ligands alters the conformation of the transporter, which increases the affinity of the UIC2 antibody for the MDR1 protein and causes a shift in fluorescence intensity [30] and [31]. Relative MFI values of 1.8 and 1.06 were obtained when MDCKII-MDR1or MDCKII-WT cells, respectively, were pre-incubated with PSC833, in line with their role as positive and negative controls ( Fig. S2; Supplementary information). Values of 1.27 and 1.26 were calculated for Calu-3 cells at a low or high passage, respectively, while NHBE cells produced a relative MFI of 1.16 ( Fig. S2; Supplementary information), indicating the presence of a MDR1 activity in bronchial epithelial cells.