Facsdiva software

Data were analyzed using FACS Diva software (TreeStar, Ashland, OR). Specific IgE (sIgE) to Af extract levels were measured with the Thermo Fisher ImmunoCAP platform (Phadia, Thermo Fisher Scientific, Uppsala, Sweden). All the results were expressed as the basophil or lymphocyte stimulation index, which is the ratio between level of activation with the allergen and level of activation with reaction buffer, with a threshold of 2. Statistical analysis was performed with the R statistical software (14 ). A correlation matrix was calculated using Pearson's correlation. Mean responses of each group were compared via the Student or Kruskal–Wallis test, a two-sided p < 0.05 was statistically significant.

Flow-cytometric analysis was performed to characterize myeloid dendritic cell (mDC) and monocyte (Mo) frequencies in PBMCs. All antibodies were purchased from BD Biosciences (San Jose, CA). Cells were stained according to BD protocols using the following mouse anti-human antibodies: CD3 (clone SP34-2), CD14 (clone M5E2), CD16 (clone 3G8), CD20 (clone 2H7), CD33 (clone P67.6), HLA-DR (clone G46.6), and CD11c (clone S-HCL-3). MDC frequencies were reported as percentage of mononuclear cells (MNC). Monocytes were further defined by gating as traditional monocytes (CD14⁺⁺CD16⁻), inflammatory monocytes (CD14⁺⁺CD16⁺) and patrolling monocytes (CD14^dim CD16⁺⁺) (see Additional file 1: Figure S1). Samples were acquired on the LSR11 (BD; San Jose, CA) using FACS DIVA software and analysed with FlowJo (TreeStar, Inc., Ashland, OR).

Flow cytometry was used to determine MP origin using a custom-built BD FACSAria II (BD Biosciences, CA). Forward scatter area (FSC-A) and side scatter area (SSC-A) were set to log scale and MPs were gated based on their FSC-A/SSC-A profile and in relation to platelets in fresh plasma. MP pellets were resuspended in 1× 0.22 μm-filtered annexin V binding buffer (BD Biosciences) and 100 μl of this was used for staining. MPs were stained in the dark (15 min, room temperature) with annexin V-FITC (1.57 μg/ml), CD41-PE-Cy5 (0.12 μg/ml), CD11b-PE-Cy7 (7.9 μg/ml), CD144-APC (4.1 μg/ml), and CD235a-PB (7.7 μg/ml) as markers of MPs, platelets, monocytes, endothelial cells, and erythrocytes, respectively (all antibodies from BioLegend, CA). Data were exported from the FACSDiva™ software (version 6) and subsequently analyzed in FlowJo software (version 9.6.4; Tree Star Inc., OR).

Immune cell profiling of frozen PBMC was performed by flow cytometry in the same 40 participants as analyzed for the PNA assay. Cryopreserved PBMCs were thawed, washed, counted and stained using a cocktail of monoclonal antibodies conjugated to different fluorochromes (BD Biosciences, San Jose, CA, USA) for specific cell surface markers (Supplementary Table S1). At least 500,000 events per sample were acquired on a BD FACSCanto™ (BD Biosciences, San Jose, CA, USA). Acquired data were analyzed using FACS DIVA software (Tree Star, Inc., Ashland, OR, USA). The different cell populations were identified based on forward- and side-scatter characteristics and cell-specific surface receptors. At least 50,000 lymphocyte-gated cells were analyzed for T- and B-cell assessment. Supplementary Figure S2 shows the process of gating and obtaining profiles of immune cells.

Prior to staining and flow cytometry analysis, the EV suspensions were treated with 1 mg ml^‐1 DNase I (Roche) in a reaction buffer containing 10 mM tris–HCl (pH 7.5), 2.5 mM MgCl₂ and 0.1 M CaCl₂, at 30°C for 1 h. EVs were stained for flow cytometry with 10 µg ml^‐1 FM4‐64 membrane dye and/or 16 µg ml^‐1 DAPI DNA dye for 15 min at room temperature while protected from light. A BD‐FACS Aria III flow cytometer was used for flow cytometry analysis. Particles from the EV suspension were gated and differentiated based on forward scatter (FSC), side scatter (SSC), DAPI and FM4‐64 parameters. A 405 nm laser with a 502 LP filter (510/50 nm) was used to detect DAPI signals and a 561 nm laser with 780/60 nm filter for detection FM4‐64 signals. For each sample, 50 000 (non‐background) events were analysed.
BD FACSDiva™ software was used for data acquisition and FlowJo flow cytometry analysis software (version 10; Tree Star, Ashland, OR, USA) was applied for data analysis.