Cd16 pb

We coated the Fluoresbrite YG Carboxylate Microspheres, 1 µm (Polysciences, 15702-10) with 2 mg/mL human immunoglobulins (IVIg, Gammunex-C) or 2 mg/mL human serum albumin (HSA, Sigma-Aldrich) and stored them at 4°C in a DPBS-NaCl buffer with 10 mg/mL bovine serum albumin (BSA, Santa Cruz Biotechnology). We then centrifuged the microspheres and replaced the supernatant with 50% autologous serum diluted in DPBS-NaCl buffers of various salt concentration (137, 200 or 300 mM). We vortexed the microspheres for 5 minutes and washed them with 1% BSA in DPBS. We resuspended them in hypertonic sodium-rich media (137, 200 or 300 mM). The microspheres were sonicated for 10 minutes, and vortexed for 5 minutes before immediate use. We isolated the neutrophils as described in 2.2., and resuspended them in respective DPBS-NaCl buffers (137, 200 or 300 mM). We added the final microsphere suspension to the cells and incubated them for 1 hour at 37°C and 5% CO₂. We stained the cells with CD15 APC (BioLegend, 1:150) and CD16 PB (BD Pharmingen, 1:150) for 30 minutes at 4°C and quantified the signal using the Gallios Flow Cytometer (Beckman Coulter). The analysis was performed in Kaluza Analysis 2.1 software (Beckman Coulter).

Freshly isolated and 5-day proliferated γδ T cells were membrane-stained with the following monoclonal antibodies; γδ TCR-FITC (Miltenyi), CD56-PE (BD), CD69-PE (BD), and HLA-DR-PE (BD). Propidium iodide (PI; Life Technologies) was added to exclude dead cells from phenotypic analysis. Data acquisition was performed on a FACScan multiparametric flow cytometer (BD). Phenotypic characterization of γδ T cells was examined pre- and post-expansion, using CD27-FITC (BD), CD69-FITC (BD), CD56-PE (BD), CD80-PE (BD), CD45RA-PE-Cy7 (BD), CD28-PerCP-Cy5.5 (BD), CD16-PB (BD), CD86-V450 (BD), γδ TCR-APC (Miltenyi), and HLA-DR-APC-H7 (BD). Live/Dead^® Fixable Aqua Stain was used to distinguish viable from non-viable cells. Data were acquired on a FACSAria II flow cytometer (BD). Corresponding species- and isotype-matched antibodies were used as controls.

We pre-incubated freshly isolated neutrophils at 37°C with general oxidative stress indicator 2 µM CM-H2DCFDA (ThermoFisher Scientific) or mitochondrial superoxide indicator 5 µM MitoSOX Red (ThermoFisher Scientific) for 20 or 10 minutes, respectively, and incubated them in respective DPBS-NaCl buffers (137, 200 or 300 mM) without serum upon Phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) or Pyocyanin (10 µM or 50 µM, respectively) stimulation for another 20 minutes. We stained the neutrophils with CD15 APC (BioLegend, 1:150) and CD16 PB (BD Pharmingen, 1:150) for 30 minutes at 4°C, and assessed the CM-H2DCFDA (ThermoFisher Scientific) and MitoSOX Red (ThermoFisher Scientific) fluorescence in the FL-2 channel with the Gallios Flow Cytometer (Beckman Coulter). The analysis was performed in Kaluza Analysis 2.1 software (Beckman Coulter).