Figure 2.

Exogenous monoamines mimic endogenous polyamine incorporation occurring in NETosing neutrophils. (a) Representative images of BPNH₂ incorporation into neutrophil intracellular and NET proteins upon 4-h activation with PMA. Neutrophils from healthy donors were preincubated with BPNH₂ for 1 h and then stimulated with PMA or left unstimulated for 4 h (control). (I) Shows BPNH₂ incorporation in resting neutrophils; (II) shows BPNH₂ incorporation in activated neutrophils; and (III) shows BPNH₂ incorporation into NET proteins. Upper panels show BPNH₂ (light blue) and DNA (red); lower panels show BPNH₂ staining alone. The experiment was repeated at least three times with neutrophils from independent healthy donors, with similar results. The original magnification was × 60, the scale bar represents 10 μm. (b) Detection of exogenous monoamine (BPNH₂) incorporation into cellular proteins in activated neutrophils by western blot. Neutrophils from a healthy donor were preincubated with BPNH₂ and then stimulated either with PMA or S. aureus or left unstimulated for 4 h (control). Levels of BPNH₂ incorporation into cellular proteins were determined from cell lysate with western blot. (c) Time-dependent incorporation of BPNH₂ into neutrophil cellular proteins upon activation. Neutrophils from a healthy donor were preincubated with BPNH₂ and then stimulated with PMA for 0–5 h. BPNH₂ incorporation levels were detected as in Figure 2b. (d) Effect of MDC on BPNH₂ incorporation into cellular and NET proteins. Neutrophils were preincubated with BPNH₂ or BPNH₂ and MDC followed by PMA stimulation for 4 h or left unstimulated (control). Cellular and NET proteins were isolated and BPNH₂ incorporation levels were detected as in Figure 2b