Isolation and Identification of Ara h 2-Specific B Cells

PBMCs were isolated by density-gradient centrifugation (Ficoll-Paque Plus, GE Healthcare) and cryopreserved in FBS with 10% DMSO. After the study outcomes were assessed, PBMCs (10 × 10⁶ to 25 × 10⁶ cells per sample) were thawed and washed with PBS. Ara h 2–specific B cells were selected by flow cytometry using a fluorescent natural Ara h 2–Alexa Fluor 488 multimer, as previously described (7 (link)), using CD3-APC (eBioscience, clone OKT3), CD14-APC (eBioscience, clone 61D3), CD16-APC (eBioscience, clone CB16), CD19-APC-Cy7 (BD Biosciences, clone SJ25C1), CD27-PE (BD Pharmingen, clone M-T271), CD38–Violet 421 (BD Biosciences, clone HIT2), and IgM-PE-Cy5 (BD Pharmingen, clone G20-127). Single Ara h 2–specific B cells, identified as Ara h 2⁺CD3^–CD14^–CD16^–CD19⁺CD27⁺ B cells, were index sorted into individual wells in a 96-well plate by FACS (Aria II or Fortessa, BD Biosciences) and stored at –80°C for single-cell B cell receptor (BCR) amplification. Data were analyzed using FlowJo software, version 8.8.7 (Tree Star).