Figure 1.
Tracking of platelet age cohorts in vivo. (A) Pulse-labeling scheme in C57BL/6J mice. (B) Representative image of isolated platelets from pulse-labeled mice spread on a fibrinogen-coated chamber. (C) Experimental outline depicting C57BL/6J mice pulse-labeled with X488 and X649 antibodies at 12-hour interval with repetitive blood sampling over time. (D) Representative gating strategy for flow cytometric analysis of pulse-labeled platelets in whole blood. (E) Graph showing percentage of labeled platelets (of all CD41+ platelets) over time, with 2-way repeated measures (RM) analysis of variance (ANOVA), comparison between labeled platelet groups (P = .0008), with the post hoc Šídák multiple comparisons test; bar graph depicting half-life of labeled platelets; paired t test, 2-tailed (P = .0021; n = 5). (F) Single-labeled platelet size; RM 1-way ANOVA (P < .0001); P-selectin expression over time in single-labeled platelets (RM 1-way ANOVA; P = .0289); phosphatidylserine exposure (RM 1-way ANOVA, P = .0178), with the post hoc Dunnett multiple comparisons test. (G) Scheme for pulse-labeling mice 108, 60, and 12 hours before sampling to determine platelet phenotype in different age cohorts simultaneously. (H) Graphs depicting single-labeled platelet percentage in circulation (n = 4 per group), platelet clearance rate (n = 5 per group), and platelet half-life (n = 4 per group); ordinary 1-way ANOVA for each graph, P < .0001; the post hoc Dunnett multiple comparisons test compared with the 0- to 12-hour group. (I) Platelet surface markers of single-labeled platelets: P-selectin mean fluorescence intensity (MFI; n = 4 per group), desialylation (RCA I binding MFI) relative to MFI of all platelets (n = 5 per group), and phosphatidylserine exposure measured by C1q binding (n = 4 per group); ordinary 1-way ANOVA, P = .0385, .0004, and .0063, respectively; the post hoc Dunnett multiple comparisons test compared with the 0- to 12-hour group. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; FSC-A, forward scatter; h, hours; ns, nonsignificant.; rel.RCA I, relative Ricinus Communis Agglutinin I binding; SSC-A, sideward scatter.

Tracking of platelet age cohorts in vivo. (A) Pulse-labeling scheme in C57BL/6J mice. (B) Representative image of isolated platelets from pulse-labeled mice spread on a fibrinogen-coated chamber. (C) Experimental outline depicting C57BL/6J mice pulse-labeled with X488 and X649 antibodies at 12-hour interval with repetitive blood sampling over time. (D) Representative gating strategy for flow cytometric analysis of pulse-labeled platelets in whole blood. (E) Graph showing percentage of labeled platelets (of all CD41+ platelets) over time, with 2-way repeated measures (RM) analysis of variance (ANOVA), comparison between labeled platelet groups (P = .0008), with the post hoc Šídák multiple comparisons test; bar graph depicting half-life of labeled platelets; paired t test, 2-tailed (P = .0021; n = 5). (F) Single-labeled platelet size; RM 1-way ANOVA (P < .0001); P-selectin expression over time in single-labeled platelets (RM 1-way ANOVA; P = .0289); phosphatidylserine exposure (RM 1-way ANOVA, P = .0178), with the post hoc Dunnett multiple comparisons test. (G) Scheme for pulse-labeling mice 108, 60, and 12 hours before sampling to determine platelet phenotype in different age cohorts simultaneously. (H) Graphs depicting single-labeled platelet percentage in circulation (n = 4 per group), platelet clearance rate (n = 5 per group), and platelet half-life (n = 4 per group); ordinary 1-way ANOVA for each graph, P < .0001; the post hoc Dunnett multiple comparisons test compared with the 0- to 12-hour group. (I) Platelet surface markers of single-labeled platelets: P-selectin mean fluorescence intensity (MFI; n = 4 per group), desialylation (RCA I binding MFI) relative to MFI of all platelets (n = 5 per group), and phosphatidylserine exposure measured by C1q binding (n = 4 per group); ordinary 1-way ANOVA, P = .0385, .0004, and .0063, respectively; the post hoc Dunnett multiple comparisons test compared with the 0- to 12-hour group. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; FSC-A, forward scatter; h, hours; ns, nonsignificant.; rel.RCA I, relative Ricinus Communis Agglutinin I binding; SSC-A, sideward scatter.

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