Statistics
The fraction of mast cells expressing CD63 was plotted against the logarithm of allergen concentrations. The maximal reactivity (%CD63+ mast cells) and mast cell sensitivity (EC50) were estimated by four-parameter logistic regression. The results for mast cell reactivity and sensitivity are given as median and interquartile range (IQR). The statistical significance of reactivity, sensitivity, prostaglandin D2 synthesis and soluble mediator expression was analyzed using 2way ANOVA. GraphPad Prism v5 was used for statistical calculations.
Normalized Protein Expression (NPX, Olink’s arbitrary unit in log2) values from the biomarkers that passed quality control (NPX value > +/- 0.3 compared to medium control), were used as input for differential expression analysis using the R Bioconductor packagelimma. Using this package, a model design was implemented to test for differential expression between non-activated mast cells (0 h) and activated mast cells (3 h, 6 h and 24 h respectively), while correcting for donor variability (cell line number) (16). For the analysis of the PEA assay, limma uses an empirical Bayesian method that considers the variance across all biomarkers to estimate per biomarker variance for model estimation. Contrasts were used to conduct comparisons of interest and generate p-values from the moderated t-test. P < 0.05 was considered significant.