2.6 Flow cytometry
Direct staining of thawed PBMC was performed with fluorescent allergen
tetramers and CD123-BV605 (6H6; BioLegend, San Diego, CA, US) and
IgE-FITC (polyclonal, Thermo Fisher Scientific) for 15 min at RT in the
dark. Flow cytometry on activated samples for the BAT was performed with
CD123-BV605, CD63-PE (H5C6; BD Biosciences) and either IgE-FITC or
HLA-DR-APC (L243; BioLegend). 7AAD was added to samples immediately
prior to flow cytometry to distinguish between live and dead cells
(Suppl Fig 1 ). All flow cytometry was performed across two
instruments (4-laser BD LSRII and 5-laser BD LSRFortessa X-20) with a
nearly identical set-up for the shared 4 lasers. Instrument set-up and
calibration was performed using standardized EuroFlow SOPs as described
in detail previously.37,38