We obtained blood samples from healthy donors with written informed consent and approval from the University of Birmingham Local Ethical Review Committee (ERN_07-058).
We isolated Peripheral blood mononuclear cells (PBMC) and neutrophils from blood using a two-step density gradient of Histopaque 1077 and 1119 (Sigma-Aldrich, Poole, UK). Lymphocytes were purified by panning PBMC on culture plastic for 30 minutes at 37°C to remove monocytes as previously described59 . Peripheral Blood Lymphocytes (PBL) were then counted, resuspended in M199 (Life Technologies Invitrogen Compounds, Paisley, U.K.) containing 0.15% bovine serum albumin (BSA; Sigma-Aldrich) at 1×106 cells/ml for transmigration assays, or in PBS containing 0.5% BSA and 2 mM EDTA (Sigma-Aldrich) for cell sorting. B cells were depleted from PBL by positive selection using anti-CD19 beads (Miltenyi Biotec, Surrey, UK). When B cells were reconstituted into PBL or used to generate supernatants, B cells were sorted by negative selection in order to yield untouched cells (Stemcell, Grenoble, France). Memory and naïve CD4 and CD8 T cells were isolated using negative selection kits (StemCell). Monocytes and their subsets were isolated by positive selection using CD14 and CD16 beads (Miltenyi Biotec).
We isolated Peripheral blood mononuclear cells (PBMC) and neutrophils from blood using a two-step density gradient of Histopaque 1077 and 1119 (Sigma-Aldrich, Poole, UK). Lymphocytes were purified by panning PBMC on culture plastic for 30 minutes at 37°C to remove monocytes as previously described59 . Peripheral Blood Lymphocytes (PBL) were then counted, resuspended in M199 (Life Technologies Invitrogen Compounds, Paisley, U.K.) containing 0.15% bovine serum albumin (BSA; Sigma-Aldrich) at 1×106 cells/ml for transmigration assays, or in PBS containing 0.5% BSA and 2 mM EDTA (Sigma-Aldrich) for cell sorting. B cells were depleted from PBL by positive selection using anti-CD19 beads (Miltenyi Biotec, Surrey, UK). When B cells were reconstituted into PBL or used to generate supernatants, B cells were sorted by negative selection in order to yield untouched cells (Stemcell, Grenoble, France). Memory and naïve CD4 and CD8 T cells were isolated using negative selection kits (StemCell). Monocytes and their subsets were isolated by positive selection using CD14 and CD16 beads (Miltenyi Biotec).