Color matched beads

The total ROS detection kit and MitoSOX (2.5 µM) were used according to the manufacturer’s instructions (Enzo Life Sciences and Life Technologies, NY). Lupus LDGs and control NDGs were incubated with either MitoSOX (5 µM) or mitoTracker^® green FM (100 nM) and cells were quantified by flow cytometry following manufacturer’s instructions for concentration and timing and using BD color matched beads for compensation (catalog number: 552843 BD Biosciences, San Diego, CA). Mitochondria were also analyzed by flow cytometry using TOM20 antibodies (Novus Biologicals) and MitoSOX (Life Technologies). In some experiments, neutrophils were treated with pronase (2 mg/mL, Calbiochem, San Diego, CA) or DNase (50 µg/mL, Roche) 30 minutes prior to addition of TOM20. Neutrophil activation was assessed by cell surface expression of CD66b (clone G10F5, BioLegend). Phagocytosis of 8-OHdG ICs was analyzed by flow cytometry and immunofluorescence microscopy upon incubation of neutrophils with Alexa Fluor-conjugated 8-OHdG ICs (2.5 µg/mL, pre-formed as described above) for 30 minutes. Phagocytosis index was determined as phagocytosed ICs per neutrophil and field of observation. Data were analyzed by FlowJo (Tree Star Inc, Ashland, OR). For all analyses, isotype antibodies or fluorescently labeled beads were used as negative controls.