Figure 2. Senescent erythrocytes are... | American Society of Hematology

Figure 2.

$Senescent erythrocytes are prone to hemolysis within the spleen in vivo. (A) Setup used to determine ghost formation in vivo. Mouse erythrocytes were stored in SAGM for 2 weeks (senescent erythrocytes) or freshly drawn and labeled intracellularly with calcein-AM and with the membrane dye PKH26 and transfused into recipient mice. Ghost erythrocytes were detected by flow cytometry (PKH+calceinlo). (B) Mouse organs were injected with collagenase buffer for 5 minutes after which cells and the number of transfused red blood cells (RBCs) in organs and circulation was quantified (autofluorescent−PKH+Hoechst− cells; n = 6). (C) Flow cytometry gating strategy used to detect labeled and transfused mouse RBCs in various organs and the circulation of recipient mice. Cells without a nucleus are depicted on the basis of Hoechst staining. Particularly in the spleen, the calcein signal was partially lost and PKH signal was retained. The top left histogram depicts unlabeled erythrocytes and the bottom left histogram depicts the labeled erythrocytes. In the top row, the 5 columns on the right depict representative histograms of nontransfused mice, and the lower 5 columns depict transfused mice. (D) Quantification of erythrocyte ghosts in the organs and circulation of transfused mice. Ghosts were defined as the fraction of cells that are autofluorescent−PKH+Hoechst−calceinlo (n = 6). (E) Imaging flow cytometric analysis of ghost formation in the spleen of a transfused mouse. The top panel indicates intact erythrocytes (PKH+calcein+) and the bottom panel depicts erythrocyte ghosts (PKH+calcein−). White lines mark cropping lines for each image because the output files were too large. (F) Live imaging of mouse spleen by 2-photon microscopy 2 hours after erythrocyte transfusion. Arrows mark PKH+calcein+ cells that become single positive roughly 2 hours after transfusion (lower right panel). (G-H) Hemopexin and haptoglobin concentrations in mouse organ and circulation-derived plasma was determined by enzyme-linked immunosorbent assay (n = 7, mean ± SD). *P < .05; **P < .01; ***P < .001; ****P < .0001. AU, arbitrary units.$

Senescent erythrocytes are prone to hemolysis within the spleen in vivo. (A) Setup used to determine ghost formation in vivo. Mouse erythrocytes were stored in SAGM for 2 weeks (senescent erythrocytes) or freshly drawn and labeled intracellularly with calcein-AM and with the membrane dye PKH26 and transfused into recipient mice. Ghost erythrocytes were detected by flow cytometry (PKH⁺calcein^lo). (B) Mouse organs were injected with collagenase buffer for 5 minutes after which cells and the number of transfused red blood cells (RBCs) in organs and circulation was quantified (autofluorescent⁻PKH⁺Hoechst⁻ cells; n = 6). (C) Flow cytometry gating strategy used to detect labeled and transfused mouse RBCs in various organs and the circulation of recipient mice. Cells without a nucleus are depicted on the basis of Hoechst staining. Particularly in the spleen, the calcein signal was partially lost and PKH signal was retained. The top left histogram depicts unlabeled erythrocytes and the bottom left histogram depicts the labeled erythrocytes. In the top row, the 5 columns on the right depict representative histograms of nontransfused mice, and the lower 5 columns depict transfused mice. (D) Quantification of erythrocyte ghosts in the organs and circulation of transfused mice. Ghosts were defined as the fraction of cells that are autofluorescent⁻PKH⁺Hoechst⁻calcein^lo (n = 6). (E) Imaging flow cytometric analysis of ghost formation in the spleen of a transfused mouse. The top panel indicates intact erythrocytes (PKH⁺calcein⁺) and the bottom panel depicts erythrocyte ghosts (PKH⁺calcein⁻). White lines mark cropping lines for each image because the output files were too large. (F) Live imaging of mouse spleen by 2-photon microscopy 2 hours after erythrocyte transfusion. Arrows mark PKH⁺calcein⁺ cells that become single positive roughly 2 hours after transfusion (lower right panel). (G-H) Hemopexin and haptoglobin concentrations in mouse organ and circulation-derived plasma was determined by enzyme-linked immunosorbent assay (n = 7, mean ± SD). *P < .05; **P < .01; ***P < .001; ****P < .0001. AU, arbitrary units.

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