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).