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.