Isolation and Profiling of Immune Cell Subsets

Blood samples were processed as described.12 (link) In brief, PBMC were obtained using density gradient centrifugation. Half underwent sequential positive selection using CD14+ and CD4+ microbeads (Miltenyi) to yield monocytes and CD4+ T cells. The remainder underwent positive selection using CD19+ and CD8+ microbeads (Miltenyi) to yield CD8+ T cells. Neutrophils were isolated from the red cell pellet by lysis followed by CD16+ microbead (Miltenyi) selection. Separation purities were monitored using flow cytometry as previously reported.10 (link) Lysed samples were kept in RLT buffer (Qiagen) at −80°C until required, and then RNA was extracted using the AllPrep Mini kit (Qiagen) and hybridised to Affymetrix HuGene 1.1 microarrays according to the manufacturer's protocols. Singaporean samples were processed in Singapore using the same protocol and then shipped to Cambridge as lysed samples in RLT buffer, with comparable separation purities and RNA quality.