Complete edta free protease inhibitors cocktail

Complete HL-5 and defined SIH (-arginine; -lysine) growth media and unlabelled L-arginine and L-lysine were from Formedium. Labelled K8 L-lysine and R6 L-arginine were from Cambridge Isotope Laboratories. Complete EDTA-free Protease Inhibitors cocktail was from Roche. Turbo DNase was from Ambion. Novex NuPAGE 4-12% gradient bis-tris gels with MOPS SDS running buffer and Novex colloidal blue staining kit were from Invitrogen. Trypsin Gold was from Promega. LavaPep peptide quantification kit was from Gel Company. ZipTip C-18 peptide purification microcolumns and high fidelity KOD hot start DNA polymerase were from Merck Millipore. Restriction enzymes, T4 DNA ligase and Antarctic Phosphatase were from New England Biolabs. Plasmid miniprep kit and PCR clean-up kit were from Qiagen. Krypton fluorescent protein stain and Nunc Lab-Tek chambered cover glass plates for cell imaging were from Thermo Scientific. All the other reagents were obtained from Sigma Aldrich.

Fourteen-day-old antibiotic-treated and drinking water– fed C57BL/6NCrl littermates were sensitized with 50 μg/ mouse Aspergillus fumigatus (Greer) and alum adjuvant (Fisher) via a single intraperitoneal injection. After 7 days, mice were challenged with intra-esophageal A fumigatus for 2 consecutive days at subsequent 2-day intervals (100 μg/mouse; 50% medium-chain triglyceride oil) over the course of another 3 weeks. Esophageal delivery was performed with an 18-gauge, 1.2-inch flexible, plastic feeding tube. The tube was perforated with an 18-gauge injection needle in 4 places at the proximal end and heat-sealed at the distal end to allow liquid to flow into the esophagus instead of the stomach. Mice were kept on antibiotics or regular autoclaved drinking water supplemented with 2% sucrose until euthanasia. To measure levels of A fumigatus–specific antibodies, enzyme-linked immunosorbent assay plates were precoated with 10 mg/mL of A fumigatus extracts (Greer) in phosphate-buffered saline (Fisher) and supplemented with complete EDTA-free protease inhibitors cocktail (Roche) for 16 hours at 4°C. Next, samples were processed according to the enzyme-linked immunosorbent assay manufacturer’s (Fisher) protocol, starting with the plate-washing step after the coating step. Serum cytokines were detected according to the enzyme-linked immunosorbent assay manufacturer’s (R&D) protocol.