Neutrophil clock controls diurnal oscillations of ischemic injury. (a) AAR in WT and mutant mice at ZT5 or ZT13 (up) and infarct sizes after correction for AAR (bottom) in the same animals subjected to MI followed by 24 h of reperfusion; n = 5 mice per group. ZT indicates the time of infarct. Analysis was performed 24 h later. (b) Differential AAR (left) or infarct size (right) between ZT5 and ZT13 in WT and mutant mice; n = 5 mice per group. (c) Representative images of infarcted hearts from WT or mutant mice infarcted at the indicated ZT. Dotted black lines highlight areas of dead myocardium. (d) Diurnal changes of CXCL12 in the plasma of WT mice; n = 4–9 mice per time point. The diurnal curves are repeated to appreciate the circadian pattern better. (e) Experimental scheme to evaluate the expression of different neutrophil surface markers at different time points after in vitro incubation of blood neutrophils from WT and CXCR4^ΔN mice. (f) Temporal transition in phenotype (shown as arrows) as determined by surface expression of CXCR4, CD62L, CXCR2, CD11b, Ly6G, and CD101 from WT blood neutrophils. (g) Kinetics of surface marker expression of blood neutrophils from control (tSNE plots and XY graphs) and CXCR4^ΔN (XY graphs) blood neutrophils at 0, 2, 4, and 6 h of in vitro incubation. n = 4–6 mice per group and time point. Data in a–c are from a single experiment. Data in d and e–g are pooled from two experiments. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, as determined by one-way ANOVA (a and b), unpaired t test analysis (a), amplitude vs. zero test (d), and two-way ANOVA (mixed model) (g). tSNE, t-distributed stochastic neighbor embedding.