Time course of human peripheral blood neutrophils incubated with (A) Texas red–labeled BSA or DQ-Green BSA for 150 minutes (data expressed as % maximum signal) and (B) DQ-Green BSA for 30 minutes followed by washing and then further incubation for 120 minutes. A and B were measured by flow cytometry (n = 3 over 2 experiments). (C) Confocal microscopy of human peripheral blood neutrophils incubated with FITC-BSA (green) and 70 kDa dextran (red) with DAPI nuclear staining in blue and red/green colocalization highlighted in yellow. Geometric mean fluorescence of LPS-treated human neutrophils incubated with (D) Texas red–labeled BSA (n = 6) or (E) DQ-Green BSA (n = 7 over 3 experiments) in normoxia or hypoxia (1% O₂) in RPMI with or without glucose (11 mM). (F) Ratio of DQ-Green to Texas red signal demonstrating breakdown efficiency in the conditions described in D and E; n = 6 over 3 experiments. Geometric mean fluorescence of BAL neutrophils from normoxic or hypoxic mice 24 hours after LPS incubated ex vivo in the corresponding oxygen tension with (G) Texas red BSA or (H) DQ-Green BSA (n = 4 over one experiment). (I) Fluorescence of cell culture supernatant from H. mTORC1 activity (proportion of phosphorylated S6 kinase) in (J) LPS-treated human peripheral blood neutrophils cultured in normoxia or hypoxia (1% O₂) with or without glucose (11 mM) (n = 7 over 4 experiments) and (K) murine BAL neutrophils isolated 24 hours after nebulized LPS (mice housed in normoxia or hypoxia); n = 8 over 2 experiments. Representative blots shown. (L) Geometric mean fluorescence of LPS-treated human neutrophils incubated with DQ-Green BSA and pretreated with MHY1485 (2 μM) or Rapamycin (50 nM); n = 4 over 2 experiments. In A and B, data represent mean ± SEM. In D–J, data represent individual values and mean ± SEM. A, B, D–F, J, and L were analyzed by ordinary 1-way ANOVA with multiple comparisons. G–I and K used the Mann-Whitney test of significance.