Mononuclear cell isolation and TLR mRNA quantification

Peripheral Blood Mononuclear Cells (PBMC) were isolated from heparinized peripheral blood by density gradient centrifugation. Human CD14 positive monocytes were isolated from PBMCs using the EasySep™ Human CD14 Positive Selection Cocktail, and neutrophils were isolated using the EasySep™ Human Neutrophil Enrichment Kit by negative selection. Initially, to ensure the robustness of the isolation procedure, a number of samples were analysed by flow cytometry to establish and confirm the purity of the monocyte and neutrophil population post selection. Next, total RNA was extracted from monocytes and neutrophils using the RNeasy isolation kit (Qiagen). RNA (1 μg per 25 μl reaction) was converted to first strand cDNA and stored at −20 °C⁴². Real-time PCR quantification was performed with DyNAmoHS SYBR Green kit (Finnzymes) using the OPTICON® system (MJ Research). For the amplification of TLRs & cytokines, in house-designed primers were used (Supplemental Table ^S1) using the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) as a reference point⁴².