Immunophenotyping by flow cytometry
Fresh whole blood samples from SSIC cohort were lysed using RBC lysis buffer (containing 155 mM NH4Cl, 10 mM KHCO3 and 0.1mM EDTA). Cells were washed using PBS and stained using Anti-IgE (MB10-5C4, Miltenyi Biotec), FcERIA (AER-37, eBioscience), CCR3 (5E8, Biolegend), CD203c (97A6, Beckman Coulter), CD123 (6H6, eBioscience), CD14 (61D3, eBioscience), CD16 (3G8, Biolegend), HLA-DR (L243, BD Biosciences), CD1c (L161, BD Biosciences). The cells were acquired on LSRFortessaTM cell analyzer (BD) and analysed using FlowJo v10 (BD). Flow cytometry data on PBMCs were generated newly for this study by using frozen PBMC samples of the SSIC cohort. Cells were thawed using pre-warmed RPMI medium (Life Technologies). Dead cells were discriminated by staining PBMCs with LIVE/DEAD Fixable Blue Dead cell stain kit (Life Technologies).  The cells were then washed using MACS buffer (0.5% BSA, 2 mM EDTA in PBS) and stained using the following antibodies:  Anti-CD49d (clone 9F10, Biolegend), Anti-CD45RB (clone MEM-55, Immunotools), Anti-CD3 (clone UCHT1, BD Biosciences), Anti-CD45RO (clone UCHL1, Biolegend), Anti-CD161 (clone HP-3G10, Biolegend), Anti-CRTH2 (clone BM16, BD Bioscience), Anti-CD4 (clone RPA-T4, BD Biosciences), Anti-CD27 (clone O323, Biolegend) and Anti-CD38 (clone HB7, BD Biosciences; O323). The cells were acquired on LSR II 5 lasers flow cytometer (BD) and analysis was performed using FlowJo v10 (BD) and unsupervised cluster analysis tools (see below).