Superb buffer

For detection of secreted CCL26, cells were seeded on 12-well plates in RPMI medium containing 5% FCS. After 24 hrs, the medium was changed, and cells were allowed to grow for an additional 24 hr prior to stimulation with IL-13 and/or NGF at a final concentration of 100 ng/ml for 24 hr. At the time of the stimulation, cells were 50% confluent. NaCl was added to the supernatant of epithelial cells to a final concentration of 100 mM prior to collection. For detecting CCL26 and NGF in biopsies, esophageal biopsies were washed twice with 0.5 ml of PBS and sonicated for 2 min in 130 μl of SuperB buffer (Covaris, Inc. Woburn, MA) with addition of EDTA and protease and phosphatase inhibitors in a microTUBE in a S220 sonicator. Sonication conditions were used as recommended by the manufacturer (10% output, 70W Peak Incident Power, 200 cycles per burst). Samples were spun down at 10,000 × g for 15 min, and supernatant was collected for analysis. The BCA protein assay was used to determine the protein concentration of each sample (Thermo Fisher Scientific Inc., Waltham, MA USA). Samples were diluted 4 times in PBS prior to ELISA. Some samples were also used for Western blotting. Human CCL26/Eotaxin-3 and NGF DuoSet ELISA kits were used according to the manufacturer’s recommendations (R&D Systems, Minneapolis, MN).