2.9.2. Immune Cell Phenotyping
Isolated PBMC were first stained with Biolegend “zombie NIR” fixable viability dye (Cat. No. 423106; San Diego, CA, USA) according to the manufacturer’s instructions for 15 min at room temperature. Cells were washed in DPBS before being stained with fluorescently labelled cell surface markers at 4 °C for 15 min. The Biolegend antibodies used were PE-Fire700 CD45 (Haematopoietic cell marker); BV605 CD3ε (T cells/natural killer T (NKT) cells); BV510 CD4 (T helper/Treg); BV650 CD8a (Cytotoxic T cells/NKT cells); BV711 CD25 (Treg/activated T cells); APC-Fire750 CD127 (Treg); PerCP CD38 (Activated NKT cells/Activated T cells/B cells); BV421 CD68 (Myeloid cells); BV570 CD56 (NK cells/NKT cells/T cells); PE-Cy7 CD16 (natural killer (NK) cells/monocytes/neutrophils); AF488 CD14 (Monocytes/neutrophils); PE-Cy5 CD15 (Neutrophils); AF647 CD11b (monocytes/macrophages); PE CD11c (monocytes/dendritic cells); BV750 HLA-DR (activated monocytes/DCs). The PBMCs were fixed in 4% formalin and then suspended in fluorescence-activated single cell sorting (FACS) buffer and analysed on a Cytek Aurora Spectral 3 laser flow cytometer (Cytek Biosciences, Fremont, CA, USA) with live spectral unmixing. Cell phenotype subpopulations were identified from Flow Cytometry Standard (FCS) files using the FlowJo software (BD Biosciences, San Jose, CA, USA).