2.3.5. PCBAT

Progenitor cell‐derived basophils were sensitized with either 20% patients' sera or with human myeloma IgE (1 μg/ml) overnight at 37°C with 5% CO₂. On the follow day, allergens extracts of different dilutions were freshly prepared from the stock using fully supplemented culture media. Sensitized PCBs were activated by incubating with serial dilutions of extracts (roasted peanut extracts or cat allergen) in the presence of serum for 30 min at 37°C. Anti‐IgE (1 μg/ml) and ‘medium only’ was included for every subject as positive and negative controls, respectively. PCBs were identified by staining the cells with CD203c⁺(FITC) and FcεRI⁺(PE‐Cy7). CD63 (PE) was used as a degranulation marker. After cells were stained with antibodies and viability dyes, Fluorescent barcoding (16‐plex) was performed using methods previously described, ¹⁹ detailed barcoding protocol can be found in this article's online repository. A minimum 5% of CD63 positive cells were required to indicate a positive PCBAT response. To depict the responsiveness of the PCBAT, we present results as AUC for CD63 expression at increasing allergen concentrations.