(A and B) ATP augments bacterial killing in a manner that is independent of phagolysosome fusion. Bacterial killing assay was performed in the presence of (A) 1-butanol or (B) bafilomycin A. Macrophages were infected with E. coli for 90 minutes and then pretreated with 1-butanol or bafilomycin A 30 minutes before ATP treatment for 5 minutes. The cells were then incubated with gentamicin for 2 hours, and intracellular bacterial CFUs were determined at the end of the gentamicin incubation period. *P < 0.05, **P < 0.01 vs. E. coli; n = 4–6. (C–H) ATP increases bacterial killing through enhancing ROS generation. (C–F) Peritoneal macrophages were infected with E. coli for 90 minutes and then treated with (C) N-acetyl-L-cysteine (NAC), (D) superoxide dismutase-polyethylene glycol (SOD-PEG), (E) 1,3-PB-ITU (nitric oxide synthase 2 inhibitor), or (F) rotenone for 30 minutes, followed by a 5-minute ATP pulse. The cells were then incubated with gentamicin for 2 hours, and intracellular CFUs were determined at the end of the incubation period. *P < 0.05, **P < 0.01 vs. E. coli treatment; ^#P < 0.05, ^##P < 0.01 vs. E. coli + ATP treatment; n = 6. (G and H) Measurement of (G) cellular or (H) mitochondrial ROS production. Peritoneal macrophages were incubated with (G) 25 μM 2′,7′-dichlorofluorescin diacetate (DCFDA) (cellular ROS dye) for 45 minutes or with (H) 5 μM MytoSOX dye (mitochondrial ROS dye) for 10 minutes. Thereafter, cells were incubated with E. coli for 90 minutes and then pulsed with ATP for 5 minutes. Following a 1-hour incubation with 400 ng/ml gentamicin, DCFDA- and mytosox-related fluorescence were detected using Victor² (Perkin Elmer) luminometer. *P < 0.05, **P < 0.01 vs. vehicle treatment; n = 6. Data are expressed as mean ± SEM. All results are representatives of 3 experiments. Data obtained by one-way ANOVA followed by Mann Whitney test.