A1ATM₃₈₃S-CF inhibits NET-mediated bacterial killing but does not inhibit neutrophil chemotaxis, reactive oxygen species (ROS) generation, or phagocytosis. PMNs were isolated from healthy adult donors for these experiments. All PMNs were pretreated ± vehicle, A1ATM₃₈₃S-CF (1.2 μM), nNIF (1.2 μM), or their SCR peptide controls. (A) Chemotaxis was determined by using recombinant interleukin-8 (70 pg/mL) as a chemoattractant in a modified Boyden chamber. The y-axis depicts the absolute number of neutrophils transversing the Transwell membrane/10 ± SEM. N = 3 to 5 samples per group. (B) ROS generation was determined by using the flow cytometric dihydrorhodamine assay. The y-axis depicts ROS generation (geometric mean ± SEM). N = 3 samples per group. PMN phagocytosis was determined after a 1-hour preincubation with A1ATM₃₈₃S-CF or SCR peptide control (1.2 μM) using 488 nm fluorescently labeled E coli (multiplicity of infection, 3:1). Immunocytochemistry was used to determine the number and fluorescent intensity of green fluorescent protein (GFP)-labeled E coli inside each PMN (yellow arrows). (C) Representative images of PMN phagocytosis at 1.2 μM concentrations of A1ATM₃₈₃S-CF or SCR peptide control (N = 3). Scale bars, 10 μm. (D) A1ATM₃₈₃S-CF or SCR peptide control treatment (1.2 μM). The y-axis depicts GFP fluorescence per PMN ± SEM for control and A1ATM₃₈₃S-CF or SCR peptide control–treated PMNs. N = 3 experiments with 20 to 70 individual PMNs assessed in each group. (E) We determined NET-mediated bacterial killing of a pathogenic strain of E coli by LPS-stimulated (100 ng/mL; 1 hour) PMNs ± A1ATM₃₈₃S-CF or SCR peptide control (1.2 μM). Micrococcal DNase (1850 mU/mL) was added to LPS-stimulated PMNs to break up NETs formed after LPS stimulation but before incubation with the E coli, and it served as a positive control for inhibition of NET-mediated extracellular bacterial killing. The y-axis depicts percentage of bacterial killing ± SEM for the 3 treatment groups. *P < .05.