Negative controls were subjected to routine conditions with the omission of the primary antibody. After washing, cells were exposed to either a 1:250 dilution PD-1/PD-L1 inhibitor of Alexafluor 568 goat-anti mouse IgG (ciliated cells) (Invitrogen Ltd, Paisley, UK) or a 1:100 dilution of FITC 488 goat anti-rabbit IgG (goblet cells) (Abcam, Cambridge, UK) for 1 h at 4 °C in the dark. Cells were once again washed three times in PBS

and the membrane was then cut out using a scalpel and mounted on a microscope slide using Vectashield with DAPI (Vector Laboratories, Peterborough, UK). Fluorescent images were viewed on a Leica SP5 confocal DMI 6000 inverted microscope equipped with a krypton-argon laser as the source for the ion beam using a x40 oil immersion objective (numerical aperture 1.25). Images were captured and viewed using LAS AF (Leica)

acquisition software. TEER was measured on days 7, 14 and 21 to ensure the formation and integrity of tight junctions between cells in the epithelium using an EVOM metre (World Precision Instruments, FL, USA) [36]. Cultures were washed with PBS and trypsinised with cytospin slides being subsequently made by spinning 5×104 cells/slide. Goblet cells and ciliated cells were detected using a 1:200 dilution of mouse monoclonal antibody against MUC5AC (Abcam, Cambridge, UK) and a 1:700 dilution selleck of mouse monoclonal antibody against acetylated alpha tubulin (Abcam, Cambridge,

UK), respectively, for 2 h at room temperature. Negative controls were subjected to routine conditions with the omission of the primary antibody. Specific binding was detected by a micro-polymer of active peroxidase coupled to anti-mouse IgG secondary antibodies Sulfite dehydrogenase (ImmPress™ Universal Reagent, Vector Laboratories, Peterborough, UK). These slides were stained using DAB peroxidase substrate kit (Vector Laboratories, Peterborough, UK) and counterstained with haematoxylin (Sigma-Aldrich, Dorset, UK). Slides (n=4 per patient per treatment) were mounted using DPX (D&H, Belfast, UK) and viewed under a light microscope. The numbers of positive cells for each stain are represented as a mean % of the total number of cells counted per slide. 1000 cells per slide were counted. WD-PBECs harvested for RNA extraction were detached by trypsinisation from the membrane and stored in RNAlater (Applied Biosystems, Warrington, UK) to stabilise the cellular RNA profile. Total RNA was extracted from stabilized cells using an RNeasy Mini kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions and quantified on a spectrophotometer. The transcription of total mRNA to cDNA was performed using First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche, UK) according to the manufacturer’s protocol.