(A–G) Murine inflammatory BAL neutrophils were isolated from WT (white bars) and myeloid-specific Phd2^–/– (black bars) mice 24 hours after in vivo LPS (3 mg) challenge and studied either at point of isolation (A–C) or following aging in culture for 6 hours (D–G). (A and D) Chemotaxis. Chemotaxis to KC (100 nM) was determined using neuroprobe chambers. (B and E). Respiratory burst. Change in DCF emission, an indicator of cellular oxidative stress, was quantified by flow following 30 minutes stimulation with fMLP (10 μM). (C and F) Inner mitochondrial membrane potential. FL2 geometric mean fluorescence with tetramethylrhodamine, methyl ester (TMRM) uptake was quantified by flow cytometry in the presence or absence of CCCP (10 μM). CCCP was used as a negative control for the TMRM measure of inner mitochondrial membrane potential and works by collapsing the proton gradient. TMRM is a cell-permeant cationic fluorescent dye that accumulates in active mitochondria and is distributed throughout the cytosol when mitochondrial membrane potential collapses. (G) Mitochondrial ROS. MitoSOX fluorescent emission was quantified by flow cytometry in the presence or absence of CCCP (10 μM). (H) Inflammatory BAL neutrophil bacterial phagocytosis. Alexa Fluor 488–conjugated E. coli BioParticles (MOI 5:1) were administered to inflammatory BAL neutrophil for 1 hour. (I and J) Apoptosis. Peripheral blood neutrophils were isolated from WT (white bars) and knockout Phd2^–/– (black bars) mice and cultured in the presence/absence of LPS (100 ng/ml) in normoxia (19 kPa, 21% O₂) or hypoxia (3 kPa, 3% O₂) for 5 (I) or 9 (J) hours and apoptosis assessed by morphology. (K) Phd2^fl/fl MRP8-Cre^+/– (MRP8;Phd2^fl/fl) and WT control mice were nebulized with LPS (3 mg) and cells harvested by BAL for total cell counts and neutrophil differential counts. Data represent mean ± SEM, n = 4. MFI, mean fluorescence intensity. P values obtained via 2-way ANOVA (A, D, I, J) and unpaired t test (G and K).