Trafficking of aged neutrophils during inflammation. Employing multichannel flow cytometry, aged and nonaged neutrophils isolated from WT mice were differentiated by pulse labeling with BrdU as detailed in “Methods.” (A) Gating strategy and results for the expression of CXCR4 and L-selectin/CD62L on aged (BrdU^neg) and nonaged (BrdU^pos) neutrophils from the peripheral blood of WT mice upon exposure to PBS or LPS (mean ± SEM for n = 4; ^#P < .05 vs nonaged neutrophils). (B) Results for numbers of aged and nonaged neutrophils in bone marrow, blood, and lysates of different organs of WT mice upon IP injection of PBS or LPS (mean ± SEM for n = 5 ; ^#P < .05 vs PBS). (C) Using multichannel in vivo microscopy on the cremaster muscle of WT recipient mice, recruitment dynamics of adoptively transferred fluorescence-labeled neutrophils isolated from WT or P-selectin/CD62P-deficient (SELP^−/−) donor mice were analyzed upon stimulation with LPS. A representative in vivo microscopy image shows aged neutrophils in red and nonaged neutrophils in green (bar represents 20 µm). (D) Results for numbers of intravascularly rolling, adherent, and transmigrated aged or nonaged neutrophils (mean ± SEM for n = 4; ^#P < .05 vs nonaged neutrophils). FSC, forward scatter; HPFs, high-power fields; MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; SEM, standard error of the mean; SSC, side scatter.