PKA activation regulates neutrophil survival. Percoll-purified neutrophils were cultured with media or (A) N6/8-AHA at concentrations of 10, 50, and 100 or (B) N6-MB-cAMP at concentrations of 500 and 1000 μM for 20 hours. (C) Neutrophils were pretreated with media (red bars) or Rp-8-Br-cAMPS (0.7 mM) (blue bar) for 30 minutes prior to the addition of N6/8-AHA (100 μM) for an additional 20 hours. (D) Neutrophils were pretreated with media (red bars) or LY294002 (10 μM) for 30 minutes (blue bars) and cultured for an additional 20 hours with GM-CSF (50 U/mL) or N6/8-AHA (100 μM). (A-D) Apoptosis was determined by light microscopy. Charts show means ± SEMs percentage apoptosis from (C) 3, (A-B) 4, or (D) 5 independent experiments. Statistical analyses were carried out by ANOVA with Bonferroni posttest and significant differences are indicated by *P < .05, **P < .01, and ***P < .001, where treated populations were compared with media control, or as indicated by lines. (E) Neutrophils were aged in culture, and RNA was isolated at time points of 1, 4, and 6 hours. In selected experiments, neutrophils were cultured with dbcAMP for 4 and 20 hours, and RNA was made at 0, 4, and 20 hours. (E-G) NR4A2/3 expression was determined by qPCR. Charts show the fold change from (E) 1-hour media or (F-G) 0-hour control, where NR4A expression was normalized to the GAPDH loading control. Each panel shows data from 3 independent experiments.