Human il 33 duoset elisa kit

We used MesoScale Discovery IL-33 V-Plex assay kit (product code K152XBD-1) and human IL-33 Duoset ELISA kit (DY3625, R&D systems) according to manufacturer’s instructions. Measurements were performed blindly by the Core Biochemical Assay lab at Addenbrookes Hospital, the University of Cambridge, Cambridge, UK.

CFTRdelF508 cells were cultured in purecol-coated 6 well plates, grown to 100% confluence and synchronized with CnT-BM.1 as described in Section Cell Culture. Cells were then pre-incubated in the presence of different kinase inhibitors or DMSO (vehicle) and infected with Pseudomonas aeruginosa (MOI of 2) for 6 h. Supernatants were then harvested and stored at −80°C. Cytoplasmic proteins were obtained with the Active Motif nuclear/cytoplasmic extract kit. Briefly, following infection, cells were washed twice with ice-cold PBS containing phosphatase inhibitors, detached, and transferred to a pre-chilled microcentrifuge tube. Cells were then precipitated by centrifugation, supernatant was aspirated and the cell pellet was resuspended in 1X hypotonic buffer. After 15 min on ice, each sample was vortexed for 10 s at the highest setting. Cell debris were precipitated by centrifugation for 2 min at 13,000 × g at 4°C and supernatants containing cytoplasmic proteins were transferred to pre-chilled microcentrifuge tubes and stored at −80°C. IL-33 protein levels were measured by ELISA with the human IL-33 DuoSet Elisa kit (R&D systems cat#DY3625-05).