pPMNs are alive. (A) Human blood was labeled with eFluor506 prior to fixation and samples were otherwise processed and analyzed by multicolor flow cytometry as described throughout the study. Expression of the viability marker was assessed on gated rsPMN (red, CD66a^low/CD63^low) and pPMN (black, CD66a^hi/CD63^hi) populations. FMO control and heat-killed cells are shown. (B) Fresh mouse blood was incubated with eFlour506 fixable viability dye. Blood was washed prior to fixation and further labeling, and analyzed by multicolor flow cytometry. FMO and heat-killed (5 minutes at 64°C) controls are shown. (C) pHrodo E coli BioParticles were incubated with human blood at 37°C, fixed, and analyzed by flow cytometry. rsPMNs (red) and pPMNs (black) had similar percentages of pHrodo⁺ cells (D) and geometric mean fluorescent intensity (gMFI) (E) at each time point. Paired comparisons were performed at each time point by 2-way ANOVA with a post hoc Bonferroni correction. (F) CellRox reagent was incubated with human blood at 37°C in the presence or absence (Cnt) of TNF-α or fMLF, fixed and analyzed by flow cytometry. rsPMNs (red) and pPMNs (black) had similar increases of the percentage of CellRox⁺ cells in response to stimulation (G). Two-way ANOVA was performed with a post hoc Bonferroni correction. rsPMN and pPMN values were compared under each condition. ns, not significant.