Rat basophilic leukemia cell-based mediator-release assay

Human FcϵRI transfected rat basophilic leukemia (RBL) cells (RS-ATL8) (50) were cultivated in Minimal Essential Medium (Gibco, Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin-streptomycin (Gibco), 0.2 mg/ml geneticin (Life Technologies, Carlsbad, CA, USA) and 0.2 mg/ml hygromycin B (Life Technologies). Cells (1.5x10⁵/well) were seeded in sterile, transparent, flat-bottomed 96-well cell culture plates (Costar, Corning Incorporated, Corning, NY, USA) and sensitized with allergic patient’s sera (1:10 diluted in medium) or with medium alone overnight at 37°C. After washing cells with Tyrod’s buffer (Tyrode’s salt (Sigma Aldrich) 24 nM NaHCO₃ and 0.1% (w/v) BSA in double distilled water) to remove unbound IgE antibodies, IgE-loaded cells were incubated with different allergen concentrations of Bet v 1, Aln g 1, Cor a 1, and Mal d 1 in Buffer B (Tyrode’s salt, 24 nM NaHCO₃ and 0.1% (w/v) BSA in 50% deuterium oxide) for one hour at 37°C to define the concentration range that causes ß-hexosaminidase release between background level and maximal response for each individual serum. To measure spontaneous release, either IgE-loaded cells were incubated with Buffer B only, or IgE-non-sensitized cells were incubated with the allergen only. Total ß-hexosaminidase release (100%) was induced with 1% (v/v) TritonX100 (Sigma-Aldrich) to lyse non-sensitized cells (data not shown).

Based on these preliminary experiments, IgE-loaded cells were exposed to three serial dilutions of allergens that were pre-incubated with Nb32ILZ, Nb32 or for control purposes with Buffer B only. In detail, allergens and Nb32ILZ or Nb32 were diluted in Buffer B, mixed 1:1 to reach final concentrations of 0.2 pM – 125 pM (allergens) and 0.25 µM – 6.25 µM (Nb32ILZ/Nb32) (to ensure an excess of nanobodies), and incubated in microplates (Greiner, Austria) for two hours at room temperature before loading them on IgE sensitized cells.

To determine the background release, either IgE-loaded cells were incubated with Buffer B only (representing the baseline) or IgE- non-sensitized cells were exposed to the individual highest allergen concentration (as shown in Supplementary Table S4 and Supplementary Table S5 ) used for each serum, e.g., Patient 1: 125pM +/- Nb32ILZ. Total β-hexosaminidase release (100%) was defined as described above. Plates were centrifuged and cell supernatants were transferred to a fresh 96-well microplate (Greiner) and mixed 1:1 with 0.16 mM 4-methylumbelliferyl N-acetyl-β-D-galactosaminide (Sigma-Aldrich) in 0.1 M citric acid buffer (pH 4.2). After one hour of incubation, glycine buffer (pH 10.7) was added to stop the reaction, and β-hexosaminidase release was measured by fluorescence (excitation wavelength: 360nm; emission wavelength: 465 nm) with a TECAN infinite M200 pro plate reader. Allergen-specific releases are given as percentages of the total mediator content (51). All results are shown as means of technical triplicates and error bars indicate standard deviation (SD).