The intracellular peptidoglycan sensor Nod1 is necessary and sufficient to mediate the microbial influence on phagocyte lifespan. Quantification of cell survival ex vivo (Annexin V⁻, % Viable) among neutrophils (PMNs) and IMs from the bone marrow (A, 24 hours) and spleens (B, 6 hours) of conventional WT, Nod1^−/−, NEO-treated Nod1^−/−, Nod2Tlr2^−/−, and Tlr4^−/− mice. Cellular lifespan was measured similarly for phagocytes obtained from CNV- and NEO-treated mice after IP injection of the Nod1 ligand C12-iE-DAP (Ligand), with 100 μg doses given 12 and 2 hours before euthanasia. (C) BrdU pulse-chase assays. BrdU⁺ frequency among PMNs and IMs from the bloodstream of CNV (black), NEO (gray), and Nod1^−/− mice (open), measured on days 2 to 5 after systemic BrdU administration. Mice received injections of vehicle (1% dimethyl sulfoxide in PBS, solid curves) or 100 μg C12-iE-DAP (Ligand, hashed curves) on days 2 and 3 after injection of BrdU. (D) Rate constants (k, day⁻¹) for 1-phase exponential decay quantified from BrdU⁺ frequencies depicted in panel C. Data presented as mean ± SEM with ≥5 mice per group. (E) Schematic for competitive neutrophil adoptive transfer assay. (F) Competitive adoptive transfer of eF450-labeled WT PMNs and CFSE-labeled Nod1^−/− PMNs into WT (black circles) or Nod1^−/− (open circles) recipient mice. Data depict the fraction Nod1^−/− among labeled PMNs 4 hours after standard transfer. (G) Competitive adoptive transfer of CFSE-labeled WT PMNs and eF450-labeled Nod1^−/− PMNs (dye-switch control). 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.