Figure 1.
Identification and characterization of P-RBC complexes in chronologically aged RBC. (A) Mice were pulse-labeled with NHS-biotin or CFSE (IV delivery), and blood samples collected from the same mice after several intervals were analyzed by flow cytometry using the method depicted. Proportions of total RBCs (ter119+) labeled with and without biotin or CFSE were quantified and gated separately, and each population analyzed for proportions of P-RBC complexes (CD41+). Each row of panels shows the proportions of biotin-labeled and unlabeled RBC and complexes in the same mouse 22 and 52 days after NHS-biotin. (B-C) Proportions of labeled and unlabeled RBCs (top, circles) and labeled and unlabeled P-RBC complexes (bottom, squares) determined at indicated times after NHS-biotin (B) (7 mice and 2 independent experiments) or CFSE (C) (6 mice). Symbols and bars indicate means and standard error of the mean (SEM). Comparisons shown between labeled and unlabeled complexes at indicated time points using 2-way analysis of variance (ANOVA) after Tukey correction for multiple testing (ns; P > .05). (D) Photomicrographs of P-RBC complexes identified in NHS-biotin–injected mice using imaging flow cytometry analysis. Single platelets are observed physically connected to RBCs and coating of RBC with platelet material (event 45 649, inset). (E-F) Levels of annexin V and FasR, respectively, on biotin-labeled and unlabeled RBCs (circles, left panels) and biotin-labeled and unlabeled P-RBC complexes (squares, right panels) at indicated times after NHS-biotin injection. Comparisons shown between labeled and unlabeled cells or complexes using 2-way ANOVA, and between labeled cells and labeled complexes using 3-way ANOVA, after Sidak correction for multiple testing. (G) Mean proportions of platelets and P-RBC labeled with anti-GPIbβ-DyLight649 (X649 positive) in blood samples from 4 mice collected at the indicated times after X649 administration (IV), compared using 2-way ANOVA. Error bars indicate SD. (H) Levels of P-selectin and high-affinity αIIbβ3 integrin on RBCs and P-RBC complexes in blood samples from 5 mice, along with levels measured in the absence of staining antibody (FMO) and in platelets either untreated or treated with 10 μg/mL collagen, as respective negative and positive stain controls. Comparisons indicated using 1-way ANOVA. dp, days past treatment; FMO, fluorescence minus one control; hp, hours past treatment; ns, not significant P > .05; SB, speed bead calibration bead; SD, standard deviation.

Identification and characterization of P-RBC complexes in chronologically aged RBC. (A) Mice were pulse-labeled with NHS-biotin or CFSE (IV delivery), and blood samples collected from the same mice after several intervals were analyzed by flow cytometry using the method depicted. Proportions of total RBCs (ter119+) labeled with and without biotin or CFSE were quantified and gated separately, and each population analyzed for proportions of P-RBC complexes (CD41+). Each row of panels shows the proportions of biotin-labeled and unlabeled RBC and complexes in the same mouse 22 and 52 days after NHS-biotin. (B-C) Proportions of labeled and unlabeled RBCs (top, circles) and labeled and unlabeled P-RBC complexes (bottom, squares) determined at indicated times after NHS-biotin (B) (7 mice and 2 independent experiments) or CFSE (C) (6 mice). Symbols and bars indicate means and standard error of the mean (SEM). Comparisons shown between labeled and unlabeled complexes at indicated time points using 2-way analysis of variance (ANOVA) after Tukey correction for multiple testing (ns; P > .05). (D) Photomicrographs of P-RBC complexes identified in NHS-biotin–injected mice using imaging flow cytometry analysis. Single platelets are observed physically connected to RBCs and coating of RBC with platelet material (event 45 649, inset). (E-F) Levels of annexin V and FasR, respectively, on biotin-labeled and unlabeled RBCs (circles, left panels) and biotin-labeled and unlabeled P-RBC complexes (squares, right panels) at indicated times after NHS-biotin injection. Comparisons shown between labeled and unlabeled cells or complexes using 2-way ANOVA, and between labeled cells and labeled complexes using 3-way ANOVA, after Sidak correction for multiple testing. (G) Mean proportions of platelets and P-RBC labeled with anti-GPIbβ-DyLight649 (X649 positive) in blood samples from 4 mice collected at the indicated times after X649 administration (IV), compared using 2-way ANOVA. Error bars indicate SD. (H) Levels of P-selectin and high-affinity αIIbβ3 integrin on RBCs and P-RBC complexes in blood samples from 5 mice, along with levels measured in the absence of staining antibody (FMO) and in platelets either untreated or treated with 10 μg/mL collagen, as respective negative and positive stain controls. Comparisons indicated using 1-way ANOVA. dp, days past treatment; FMO, fluorescence minus one control; hp, hours past treatment; ns, not significant P > .05; SB, speed bead calibration bead; SD, standard deviation.

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