Cd16 apc h7

Anti-human CD66bFITC, CD3, CD19, CD14, CD163,CD138, CD56, CD15 all PE conjugated, HLADRV450, CD45V500, CD16APC-H7, CD14PE-Cy7, CD38PE-Cy7, CD 206APC were obtained (BD Pharmingen, Stockholm, Sweden). CD34PE, CD235aPE, CD11bPerCp-eFlour710 (eBioscience, San Diego, CA, USA), CD192APC, and CX3CR1AlexaFlour647 were from BioLegend, San Diego, CA, USA. Human FcReceptor binding inhibitor, 7AAD, and Annexin V-Alexa647, were obtained from eBioscience, San Diego, CA, USA, Sigma–Aldrich, (St. Louis, MO, USA), and Molecular Probes, Eugene, OR, USA, respectively.

Whole blood and bone marrow samples were lysed using 0.84% NH₄Cl and stained with the following antibodies: CD14—PerCP Cy5.5 (clone: M5E2, BD), CD80—FITC (Santa Cruz Biotechnology, Dallas, TX, United States), Siglec8—PE (clone: 7C9, BioLegend, San Diego, CA, United States), CD16—APC H7 (clone: 3G8, BD), CD66b—Alexa flour 700 (clone: G10F5, BioLegend), CD184—APC (clone: 12G5, eBioscience, San Diego, CA, United States), CD11b—BV785 (clone: ICRF44, BioLegend), CD62L—BV650 (clone: DREG-56, BioLegend), CD193—V510 (clone: 5 E8, BD), CD45—V450 (clone: 2D1, BD), HLA-DR—PE Cy7 (clone: L243, BD), and CD69—PE/DAZZLE (clone: FN50, BioLegend). The samples were then analyzed on FACS Aria Fusion (BD), for gating strategy see supplement figure 1. Single cells were gated, whereupon neutrophils were characterized as CD45⁺CD14⁻CD193⁻ cells and then further divided into mature, immature, and CD11b⁻ immature neutrophils based on their expression of CD11b and CD62L. When investigating the level of activation before and after isolation, as well as when analyzing the purity, the same antibody panel was used but the analysis was performed on Cytoflex (Beckman Coulter, Brea, CA, United States). All data was then analyzed using the Kaluza software (BD).

For the differentiation and polarization of isolated human monocytes into M1 and M2 macrophages, cells were cultured for 7 days at 37 °C in a 5% CO₂ atmosphere in complete medium composed of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution in the presence or absence of different cytokines. Monocytes were seeded in 96- or 6-well culture plates at 2 × 10⁵ cells/well or 1 × 10⁶ cells/well, respectively, and the following cytokines were added for 7 days: 50 ng/mL GM-CSF (for M1 polarization) or 50 ng/mL M-CSF (for M2 polarization). At day 6, 100 ng/mL LPS was added to M1 polarized macrophages and 20 ng/mL IL-4 was added to M2 polarized macrophages. Seven days later, cells were detached from plates and stained with a cocktail of conjugated antibodies as follows: 10 µL CD14-FITC, 5 µL CD16-APC-H7, 5 µL CD80-PE-Cy5, 5 µL CD163-APC, and 5 µL CD200R-PE from BD Biosciences. Data acquisition was performed on 10,000 cells with constant PMT values on an FACS Canto II cytometer (BD Biosciences). Data analysis was carried out using Diva software (BD FACSDiva v9.0software, BD).

ROS production by neutrophils and monocytes was analyzed by incubating whole blood with dihydrorhodamine 123 (DHR) and catalase in the presence or absence of PMA. Red blood cells were lysed with BD FACS lysing solution (#349,202, BD) and stained with the following antibodies: CD16 APC-H7 (#561,306, BD), CD66b V450 (#561,649, BD), and CD45 V500-C (#655,873, BD). Analysis was performed on a BD FACSCanto II (BD). H₂O₂ production by MDMs with and without phorbol 12 myristate 13 acetate (PMA) stimulation (400 ng/mL) was assessed with the Amplex Red Kit (#A22188, Thermo Fisher Scientific) in Krebs–Ringer bicarbonate buffer (K40002, Merck). Briefly, 3 × 10⁴ cells were cultured in RPMI 1640 containing 10% FCS in 96-well flat-bottom plates. MDMs were primed with 1000 IU/mL human recombinant interferon gamma (IFN-γ) (Imukin, Boehringer Ingelheim) 16 h before the experiment. H₂O₂ release was quantified with a Victor X4 plate reader (Perkin Elmer).

The following antibodies were used for flow cytometry: CD3-PerCP-Cy5.5 (clone SK7; eBioscience; used 1:20); CD4-FITC (clone RPA-T4; BD Biosciences; used 1:20), CD4-APC (clone RPA-T4; BD Biosciences; used 1:30), CD4-BV421 (clone SK3, Biolegend; used 1:100), CD8-BV421 (clone RPA-T8; BD Biosciences; used 1:50), CD14-APC-H7 (clone MoP9, BD Biosciences; used 1:100), CD16-APC-H7 (clone 3G8, BD Biosciences; used 1:100), CD19-FITC (clone 4G7, BD Biosciences; used 1:30), CD137-BV421 (clone 4B4-1; Biolegend; used 1:200), CD137-APC (clone 4B4-1; BD Biosciences; used 1:30), OX40-PE-Cy7 (clone Ber-ACT35, Biolegend), CD107-PE (clone H4A3, BD Biosciences; used 1:150) and PE-conjugated anti-mouse TCRβ constant domain (clone H57-597; BD Biosciences; used 1:150). The viability stain IR-Dye (Thermo Fisher, used 1:2,000) was used to identify live cells.