Depletion of neutrophils in myocardial infarction experiments. (a) Number of neutrophils in circulation in control and neutrophil-depleted mice after bleeding mice at different circadian times; n = 2 mice per time point. The diurnal curves are repeated to appreciate the circadian pattern better. (b) Gating strategy for neutrophil identification after depletion. (c) Experimental scheme for the I/R (AMI) model at two different circadian times and antibody cocktails used as control or to deplete neutrophils. ZT indicates the time of infarct. Analysis was performed 1 h and 45 min later. (d) AAR of the myocardium (left) in control and neutropenic mice at ZT5 or ZT13 and infarct sizes (right) after correction for AAR from the same animals subjected to MI and followed by 1 h of reperfusion; n = 4 mice per group. (e) Experimental scheme for evaluation of immune cell populations in the blood and heart of naïve and infarcted mice after neutrophil depletion. Analysis was performed 24 h after reperfusion. (f) Heatmap representing the numbers of the indicated immune cell populations in the blood of control and neutrophil-depleted mice at ZT5, in basal conditions and after AMI; n = 4 mice per group (for naïve) and n = 3–4 mice per group (for AMI). Data are normalized to the control isotype (iso) group for each cell type. (g) Heatmap representing the numbers of the indicated immune cell populations in the heart of control and neutrophil-depleted mice at ZT5, in basal conditions and after AMI; n = 4 mice per group (for naïve) and n = 3–4 mice per group (for AMI). Data are normalized to the control isotype (iso) group for each cell type. Data in a–d are from single experiments. Data in f and g are pooled from two experiments. Data are shown as the mean ± SEM. **P < 0.01; ***P < 0.001; ns, not significant, as determined by amplitude vs. zero test (a), two-way ANOVA (d), and unpaired t test (f and g). Neus, neutrophils; Eos, eosinophils; Macs, macrophages; Mono, monocytes.