C1q inhibits HMGB1 internalization by human monocytes. (A) Human monocytes were incubated with AF555-labeled HMGB1 with (+C1q) or without C1q (−C1q) for 30 minutes at 4°C or 37°C and stained with fluorescein isothiocyanate–anti-CD14 antibody. Imaging flow cytometry was performed on an ImageStreamX Mark II. Intensity of AF555-HMGB1 of a CD14⁺ population in each condition is shown. (B) The Internalization Wizard following membrane masking was used to quantify intracellular HMGB1. The median fluorescent intensity from 3 independent experiments is shown. ***P < .001. (C) Representative images of monocytes that internalized HMGB1 (red) without C1q (left) and in the presence of C1q (right).