Proteins, antibodies and media.
Antibody for detection of Hoxb8 MC activation: monoclonal rat anti-mouse CD107a APC and PE (clone 1D4B, BioLegend, CA, USA). Activation medium: RPMI-1640 w/ stable glutamine, 2.0 g/L NaHCO3 (Seraglob, Bioswisstec AG, Schaffhausen, Switzerland) complemented with 10% Hyclone FCS (Fisher Scientific, Hampton, NH), 100 U/mL penicillin, 100 mg/mL streptomycin (1003 penicillin/streptomycin, Gibco), 10 mM HEPES buffer solution (stock-solution 1 mol, Gibco), 1 mM sodium pyruvate (stock-solution 100 mM, 1003, Gibco), 4 mM L-glutamine (stock-solution 200 mM, 1003, Gibco), 13 nonessential amino acids (stock-solution 1003, Gibco), 30 ng/ mL mouse recombinant IL-3 (Peprotech), and 50 mM 2-mercaptoethanol (stock-solution 14.3 mol, Merck, Darmstadt, Germany). RPMIc: RPMI1640 medium AQmedia (Sigma-Aldrich, St Louis, Mo) complemented with 10% FCS Sera Pro (Pan Biotech, Aidenbach, Germany), 100 U/mL penicillin, 100 mg/mL streptomycin (1003 penicillin/streptomycin, Gibco by Sigma-Aldrich) , and 50 mM 2-mercaptoethanol (stock-solution 14.3 mol, Merck, Darmstadt, Germany). RPMIc/IL3: RPMIc plus 10% WEHI-3b conditioned medium.
Statistical analyses. SPT and sIgE measurements results of PA and NA groups were compared using Student’s unpaired t-test. The simple linear regression model was used to correlate SPT and sIgE measurements with maximal Hoxb8 MAT results. Two-way ANOVA with Sidak’s multiple comparison was used to compare BAT and Hoxb8 MAT results at individual allergen concentrations between PA and NA groups. Performance of SPT, sIgE measurements, BAT and Hoxb8 MAT was examined against the clinically validated allergic status of individual samples using receiver-operating characteristic (ROC) curves via either the R package cutpointr (version 1.1.2) to find the optimal cut point or by application of a logistic regression models in connection with the R package ROCR (version 1.0-11). GraphPad Prism (Version 10; Pad Software, La Jolla, CA, USA) and R (Version 4; R Core Team 2023) were used for statistical testing.