Cd16 pecy7 3g8

Aliquots of peripheral blood mononuclear cells (PBMC) from malaria-naive healthy individuals recruited in Melbourne were incubated with predetermined saturating concentrations of the relevant antibodies. Monocytes were gated using forward and side scatter and monocyte subsets identified with CD16 PE Cy7 (3G8, BD Biosciences) and CD14 BV510 (M5E2, Biolegend) or APC (M5E2, BD Biosciences). antibodies used were: CD16 PE Cy7 (3G8, BD Biosciences), CD32a (IV.3 biotinylated Fab fragment + streptavidin APC), CD32b (in house (MH), 63X-21 biotinylated whole IgG + streptavidin APC) [32 (link)], CD64 PerCP 5.5 (10.1, Biolegend), CD11a Alexa-488 (HI111, Biolegend), CD11b APC (ICRF44, Biolegend), activated CD11b (CBRM1/5-FITC, Biolegend), CD11c V450 (B-ly6, BD Biosciences), CD35 FITC (E11, Biolegend).

Patient blood samples were obtained at the time of presentation and processed immediately to avoid coagulation and apoptosis. Venous blood samples were drawn in pyrogen-free tubes. To obtain leukocyte suspensions, whole blood was diluted 1:1 with DPBS and 20 ml diluted blood was overlaid on a 15 ml density gradient (Ficoll-Paque Plus, density 1.077 g/ml, GE Healthcare, NJ) and centrifuged (20 min, 1600 rpm, 18 °C). The mononuclear cell interphase was carefully isolated and washed 3 times with DPBS. Cell suspensions were frozen at −80 °C until further analysis.
Cell suspensions were stained with the following antibodies (all from BD Bioscience) at a final concentration of 1:100: CD11b-V450/ICRF44, CD14-PerCP-Cy5.5/M5E2, CD16 PE-Cy7/3G8, CCR2-Alexa-647/48607, CX3CR1-FITC/2A9-1, MPO-FITC/5B8, CD162-PE/KPL-1, and CD106-PE/51-10C9. Intracellular MPO staining was performed after fixation and permeabilization (BD Cytofix/Cytoperm, BD Bioscience, San Jose, CA). Cell phenotyping was performed using a Cyan Flow Cytometer (Beckman Coulter, Fichtenhain, Germany) after appropriate compensations. Flow cytometric data were analyzed using FlowJo v.8.5.2 (Tree Star, Inc., Ashland, USA).

20 mL of the whole blood on lithium heparin anticoagulant was collected from HIV-infected individuals and HC. Neutrophils were purified by negative selection by microbeads, which allowed the removal of DCs, B cells, monocytes, macrophages, activated T cells, and activated NK cells (MACSxpress Whole Blood Neutrophil Isolation Kit; cat. 130-104-434). Residual erythrocytes were lysed with the use of 2mL ammonium chloride Lysing Reagent (BD cat. 555899) for 5 minutes. The final purity of PMN population was assessed by flow cytometry using CD14-PE (clone M5E2), CD15-FITC (MMA), and CD16-PECy7 (3G8, all from BD Pharmingen) mAbs. Flow cytometric analysis of isolated populations of cells showed that the percentage of CD15^highCD16⁺CD14^- neutrophils was >98%. The level of contaminating CD14⁺CD15⁺ monocytes was about 0.4% and CD15⁺CD16^- eosinophils was <0.1% after isolation (Figure S1A in Supplementary Material). 2x10⁶ neutrophils were incubated without stimulation, in the presence of 100ng/mL ultrapure LPS from E. coli (serotype R515, Alexis Biochemicals) in RPMI 1640 for 6 h (5%CO₂, 37°C, humid atmosphere). For DNA isolation, samples of isolated neutrophils were frozen and kept at -150°C.