Histopaque

PBMC was isolated from heparinized whole blood following standardized procedures [15 (link)]. Briefly, using sterile 10 ml disposable pipettes (Sarstedt), blood from the three 10 ml vacutainers per participant was transferred into a sterile 50 ml centrifuge tube (GBO) labeled with the participant unique identification number. An equal volume of pre-warmed (37°C) RPMI 1640 (GIBCO) was added to the blood in the falcon tube to achieve a 1:1 dilution and mixed gently. The diluted blood was layered gently onto 15 ml of Histopaque (SIGMA Lot No. H8889) without breaching the Histopaque-blood barrier in a ratio of 2:1 for blood and Histopaque. Both the blood and Histopaque were used at room temperature. The tubes were centrifuged at 800 g for 30 min at room temperature with the brake off. The milky-white ring of PBMC between the Histopaque (transparent) and plasma (yellow) was then aspirated with a sterile pastette into sterile 50 ml tubes, and washed twice with pre-warmed Hank’s Balanced Salt Solution (SIGMA Lot No. H9394). The cells were finally suspended in 1 ml of filtered growth medium for counting using 1 in 2 dilution with 0.4% Trypan blue (GIBCO: Cat. No. 15250–061).

The procedure has been described previously [11 (link)]. Briefly, mice were perfused with PBS under deep anesthesia with 3–4% isoflurane, and the brains and spleens were extracted. The brains were cut into small pieces and passed by 18-gauge and 20-gauge needles. The minced brain tissue was washed, resuspended in 25% Percoll (GE Healthcare), and centrifuged for 20 min at 500×g. The supernatants were discarded. The cell pellets were resuspended in Histopaque (Sigma) and centrifuged for 20 min at 500×g. Spleens were minced, filtered by 70-μm mesh filters, resuspended in Histopaque, and centrifuged for 20 min at 500×g. For both, enriched immune cells were recovered at the Histopaque interface. The cells were corrected and incubated with the following antibodies: PerCP-Cy5.5-conjugated anti-mouse CD11b monoclonal antibody (BD Pharmingen), phycoerythrin (PE)-Cy7-conjugated anti-mouse CD45 monoclonal antibody (BD Pharmingen), APC-conjugated anti-mouse Ly-6C monoclonal antibody (BD Pharmingen), and BV421-conjugated anti-mouse Ly-6G monoclonal antibody (BD Horizon). Isotype controls were also used. The fluorescent-labeled cells were analyzed by LSRFortessaX-20 (BD Biosciences) and BD FACSDiva software (BD Biosciences). Analysis of flow cytometry results was conducted on FlowJo software version 10.5 (BD Bioscience).

Wild-type C57BL/6, Rab27a^+/+, Rab27b^+/+, ashen Rab27a^ash/ash, Rab27b knockout (Rab27b^−/−) and Rab27a/b double knockout (Rab27DKO) mice were bred in-house and generated as described previously [21 (link),26 (link)]. All animal experiments were performed with the approval of an ethics committee and in compliance with the UK Home Office Regulations under PPL 70/7078 at the Central Biomedical Sciences of Imperial College, London, UK. Neutrophils from mouse bone marrow were purified as described previously [36 (link)]. In brief, bone marrow cells were flushed from femurs and tibias of mice with PBS and red blood cells lysed by resuspension in a solution of 0.168 M NH₄Cl, 10 mM KHCO₃ and 0.097 mM ethylenediaminetetraacetic acid (EDTA). Cells were washed once with phosphate buffered saline (PBS), resuspended in 1 mL of PBS and layered on top of a discontinuous Histopaque gradient containing 3 mL of Histopaque (Sigma-Aldrich) at 1.119 g/mL at the bottom and 3 mL of Histopaque at 1.077 g/mL on top. Cells were centrifuged for 45 min at 700 g in a swing bucket centrifuge without braking. Cells at the interface of the two layers were collected and washed twice with PBS. Typical preparations contained above 80% neutrophils as assessed by Ly6G^high staining by flow cytometry.