Carboxylate modified microspheres

Neutrophil isolation and Pc and OB assays were performed according to the procedures described in detail by Miltenburg et al. (2018b) . All samples were processed and run by the same operator who was blinded to the treatment group. Blood samples (ACD-blood) were first diluted with 1 × PBS (20 mL) at room Carboxylate-Modified Microspheres, Thermo Fisher Scientific, Mississauga, ON, Canada; 1.0 µm (488/560 nm, excitation/emission), 2% solids] in the dark for 30 min at 37°C. An aliquot free of beads (control) was simultaneously analyzed for each sample. After 30 min, 200 µL of cold 1× PBS was added to each tube and placed on ice until flow cytometry analysis. For the OB assay, 2 flow cytometry tubes, each containing 200 µL of neutrophils (1 × 10 6 cells) diluted in PBS containing 10% filtered fetal bovine serum (Invitrogen, Burlington, ON, Canada), were incubated at 37°C with 2 µL of 1 mM 2′,7′-dihydro-dichlorofluroscein-diacetate (H 2 DCFDA; Molecular Probes, Eugene, OR). After 15 min, OB stimulation was performed by adding 200 µL of 25 ng/mL PMA (Sigma-Aldrich) to 1 tube. Simultaneously, 200 µL of 1× PBS with fetal bovine serum were added to the control tube. Both samples were further incubated for 15 min and then placed on ice until flow cytometry analysis.