Antibiotic treatment corresponds with impaired phagocyte survival ex vivo. (A) Representative flow cytometry plots of neutrophils (PMNs, gated on CD45⁺CD11b⁺Ly6C⁺Ly6G⁺) obtained from bone marrow (BM) and spleens and allowed to incubate ex vivo for 0, 6, or 24 hours before staining with Annexin V. Nonapoptotic, viable cells were quantified by gating on Annexin V⁻ events (SSC, side scatter). (B) Assessment of Annexin V⁻ event frequency (% Viable) among PMNs and IMs obtained from the BM and spleens of conventional (CNV, black), broad-spectrum antibiotic-treated (ABX, open), and neomycin-treated (NEO, gray) mice following 6 or 24 hours incubation ex vivo. Steady-state frequencies (% of CD45⁺ events) of PMNs and IMs obtained from the BM (C) and spleens (D) of CNV (black circles) and germ-free mice (GF, open circles). Absolute cell numbers: BM: CNV PMNs 10.83 ± 0.37, GF PMNs 8.40 ± 0.56, CNV IMs 2.24 ± 0.04, GF IMs 2.01 ± 0.12 (all ×10⁶ cells per femur). Spleen: CNV PMNs 1.07 ± 0.12, GF PMNs 0.58 ± 0.08, CNV IMs 2.67 ± 0.33, GF IMs 1.81 ± 0.15 (all ×10⁶ cells per spleen). Survival ex vivo of PMNs and IMs from the BM (E, 24 hours) and spleens (F, 6 hours) of CNV and GF mice. Horizontal lines in scatter plots depict median values. All other data presented as mean ± SEM with ≥3 to 5 mice per group. Statistical significance was assessed by Student t test for pairwise comparisons and 1-way ANOVA with Newman-Keuls posttest for comparisons of >2 conditions. *P < .05; **P < .01; ***P < .001.