Cd14 selection beads

Whole venous blood was collected into tubes containing 1.8 mg/ml K₂EDTA (Becton Dickinson, Oxford, UK). PBMCs were isolated using Ficoll-plaque gradients (Cedarlane, Burlington, ON, Canada) as previously described [17 (link)]. Monocytes were isolated from PBMCs using CD14⁺ selection beads (Miltenyi Biotec, Bisley, UK) as per the manufacturer’s instructions before being cultured in RPMI1640 medium supplemented with 5% (v/v) fetal calf serum (PAN-Biotec, Aidenbach, Germany) and 1% (v/v) penicillin–streptomycin solution (PAA, Pasching, Austria). Cells were incubated with or without 100 ng/ml PAM₃CSK₄ (PAM3) or 10 ng/ml flagellin (Axxora, Exeter, UK), 1 ng/ml FSL-1, 10 ng/ml lipopolysaccharide (LPS), 2 μg/ml resiquimod (R-848), 2 μM ODN2006 or 2 μM ODN2216 (Invivogen, Toulouse, France) for 18 h at 37°C, 5% CO₂. The concentrations of the TLR ligands were previously determined by titrating each ligand to determine the threshold for maximal activation.

Whole venous blood was collected into tubes containing 1.8 mg/ml (K2) EDTA (Becton Dickinson, Oxford, UK) and stored at room temperature for up to 2 hours prior to cell separation. Leucocyte cones from blood donors were purchased from NHS Blood and Transplant (Tooting, UK) to determine SARM expression upon TLR activation . Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque gradients (Cedarlane, Burlington, Canada) as previously described [18 (link)]. Monocytes from PBMCs derived from whole venous blood were isolated using CD14⁺ selection beads (Miltenyi Biotec, Cologne, Germany) as per the manufacturer’s instructions. Peripheral blood monocytes from PBMCs derived from leucocyte cones were isolated by iso-osmotic Percoll gradient centrifugation, as previously described [19 ], before being cultured in RPMI1640 media supplemented with 5% (v/v) foetal calf serum and 1% (v/v) penicillin/streptomycin solution (PAA, Pasching, Austria) with or without 100 ng/ml PAM₃CSK₄ (Axxora, Nottingham, UK), 10 ng/ml lipopolysaccharide (LPS) (Axxora, Nottingham, UK) or 2 μg/ml resiquimod (R-848) (Enzo Life Sciences, Lausen, Switzerland) at 37°C, 5% CO₂. All TLR ligands were used at predetermined concentrations that induce maximal cytokine induction.