Cd16 v500

PBMCs were stained with fixable viability dye (for dead cell exclusion, eBioscience, San Diego, CA, USA) and antibodies for CD14 APC (Miltenyi Biotec), CD11c PerCPCy5.5 (Biolegend, San Diego, CA, USA), HLA-DR FITC (BD Biosciences, Franklin Lakes, NJ, USA), CD16 V500 (BD Biosciences), Tie2 PE and its respective isotype control (R&D systems). Data were acquired on a BD FACSCanto II (BD Biosciences). After excluding debris, doublets, and dead cells, cell populations (see gating strategy in Table 2) were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA). Results were expressed as percentage of positive cells.

MitoTracker green (MTG) was used to assess mitochondrial mass of leukocytes. MitoSox red was used to assess mitochondrial superoxide production of leukocytes. Generation of mitochondrial ROS (mtROS) and mitochondrial mass were evaluated using flow cytometry. Whole blood was incubated at 37°C for 1 h untreated (vehicle) or with 10 ng/mL LPS in the presence of MTG dye (1.4 μM) and MitoSox red reagent (15 μM) (ThermoFisher Scientific). Samples were stained with mAb for CD15- PECy7, CD66b-PB (BioLegend®, USA), CD14-APC (Beckman Coulter), and CD16⁻V500 (BD Biosciences, UK) for 15 min. BD lysis buffer was used to lyse red blood cells. Cells were acquired on a BD FACSCanto II flow cytometer. Fluorescence minus one (FMO) controls were used to set gates. Mitochondrial mass and superoxide were quantified based on MFI of MTG and MitoSOX red, respectively (17 (link)). Data were analyzed using FlowJo software version 10.