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Identification of Stages of Erythroid Differentiation in Bone Marrow and Erythrocyte Subpopulations in Blood Circulation that Are Preferentially Lost in Autoimmune Hemolytic Anemia in Mouse

Fig 5

Generation of Reactive Oxygen Species (ROS) in control and AIHA-induced mice.

Mice were given intraperitoneal injections of 2x108 rat erythrocytes weekly for 5–6 weeks to induce AIHA. Blood samples were collected from control and AIHA-induced mice 3 days after 5th and 6th doses of injection. Erythrocytes were stained with CM-H2DCFDA and intracellular ROS generation was determined by flow cytometry. Representative histograms showing ROS generation in control and AIHA-induced mice are shown in panel A, and the mean ROS level in whole erythrocyte population is shown in panel B (p = 0.010, ANOVA). Mouse erythrocytes were labeled with biotin in vivo by the two step biotinylation procedure (as per schedule given in Fig 3, panel A). Erythrocytes from DIB stained mice were incubated with CM-H2DCFDA and stained ex vivo with streptavidin-APC and anti-mouse CD71-PE. Erythrocytes of different age groups (reticulocytes, biotinnegative, biotinlow and biotinhigh) were gated (as in Fig 3, panels B and C) and ROS level was analyzed in each of them. ROS level in the different age cohorts of erythrocytes are given in panels C-F. ANOVA tests for each of the subgroups reveal p = 0.741 for reticulocytes (panel C), p = 0.009 for biotinnegative (panel D), p = 0.022 for biotinlow (panel E) and p = 0.005 for biotinhigh (panel F). Each bar on the graph represents mean ± SEM of observations. n = 8 control and 12 AIHA-induced mice. *p<0.05, **p<0.01 and ****p<0.001 for comparison of the groups (Student t-test).

Fig 5