Extended Data Figure 1. Phenotypic and functional characterization of aged neutrophils.

a, Flow cytometry analysis of donor neutrophil ageing after adoptive transfer into recipients. Donor neutrophils gated by CD45.1⁺ and aged neutrophils gated by CD62L^loCXCR4^hi. b, Ageing and clearance kinetics of donor neutrophils after adoptive transfer into recipients (n = 3 mice). Left y-axis, donor neutrophil number relative to the initial number of neutrophils transferred (black dash line); right y-axis, percentage of the aged subset in donor neutrophils (red line). c–d, MFIM analysis of Mac-1 activation of neutrophils harvested from WT or Selp^−/− mice, labelled by PKH26 (red) and transferred into WT recipients. Scale bar, 10 μm. e, Plasma cytokine levels in WT and CD169-DTR mice 5 days after DT treatment (n = 5 mice). f, Percentages of adherent neutrophils that capture > 8 beads in diphtheria toxin (DT)-treated WT and CD169-DTR mice (n = 8 mice). g–h, Flow cytometry analysis of surface marker expressions (g), cell size (FSC) and granularity (SSC; h; n = 7 mice) on CD62L^hi young and CD62L^lo aged neutrophils. i, CXCR4 expression levels on CD62L^hi young and CD62L^lo aged neutrophils in WT, Selp^−/−, and CD169-DTR mice (WT: n = 13 mice; Selp^−/−: n = 4 mice; CD169-DTR: n = 5 mice). Error bars, mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, data representing ≥2 independent experiments analysed with one-way ANOVA (b) or unpaired Student’s t-test (e–i).