Optimized Depletion of Immune Cells

For comparative depletion experiments, heparinized blood was divided into aliquots and incubated with Miltenyi MicroBeads at 2–8°C. Volumes and incubation times were optimized for each antigen and achieved approximately 90% depletion of the target cell [anti-CD4: 100 mcl/ml, 30 min; anti-CD8: 50 mcl/ml, 15 min; anti-CD14: 100 mcl/ml, 30 min; anti-CD15: 50 mcl/ml, 15 min; Basic (unconjugated) MicroBeads used for controls: 50 mcl/ml, 15–30 min; see Figure S1 in Supplementary Material]. Blood was diluted 1:1 with RPMI-1640 and passed through Miltenyi Biotec LS columns supported in magnets (MidiMACS Separation Unit, Miltenyi Biotec); columns had been pre-“primed” with 3 ml MACS buffer [0.5% bovine serum albumin (Sigma) and 2 mM EDTA (Sigma)], all as previously described (2 (link)). Depleted blood was collected in Universal containers. Granulocyte isolation using discontinuous Percoll gradient was performed as previously described (2 (link)). Neutrophils were rendered necrotic (when required) via heat shock at 60°C for approximately 20 min, until all cells were trypan blue (Sigma) positive by microscopy, and then allowed to cool.