Tyrode s salts

Rat basophil leukemia (RBL) RS-ATL8 cell line transfected with the human high-affinity IgE receptor (FcεRIH) [50 (link)] was kindly provided by Dr. Ryosuke Nakamura (Tokyo, Japan). Cells were plated (1.5 × 10⁵ cells/well) on 96-well sterile tissue culture plates (Corning, New York, NY, USA) added with cell culture medium MEM supplemented with 10% FCS, 0.2 mM L-Glutamine, 100 U/mL Pen/Strep 0.2 mg/mL geneticin (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) and 0.2 mg/mL hygromycin B (Invitrogen, Thermo Fischer Scientific, Waltham, MA, USA) and loaded with 1:10 diluted serum, previously heat-inactivated 25 min at 56 °C [51 (link)] and then incubated overnight at 37 °C, 5% CO₂. For this experiment, sera from seven patients positive in ImmunoCAP to ragweed profilin within our study cohort were used. After washing with Tyrod Buffer containing Tyrode’s Salts (Sigma-Aldrich, Vienna, Austria) and 1.0 g/L NaHCO₃, degranulation of RBL cells was induced upon stimulation with 6 different concentrations of rAmb a 8 and rArt v 4 (0.01–1000 ng/mL). nAmb a 1.01, produced as described [52 (link)], was used for control purposes. Buffer without allergens was used as the negative control. Results are shown as the percentage of total ß-hexosaminidase release. All measurements were performed in triplicate and represent means ± SDs.