Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis by Jing Liu, Jianhua.

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Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis by Jing Liu, Jianhua Zhang, Yelena Ginzburg, Huihui Li, Fumin Xue, Lucia De Franceschi, Joel Anne Chasis, Narla Mohandas, and Xiuli An Blood Volume 121(8):e43-e49 February 21, 2013 ©2013 by American Society of Hematology

Flow cytometric analysis and isolation of erythroblasts of wild-type mice bone marrow cells. Flow cytometric analysis and isolation of erythroblasts of wild-type mice bone marrow cells. (A) Plot of FSC versus SSC of all the cells. (B) Plot of 7AAD versus SSC of all the cells. (C) Plot of CD45CD11bGR1 versus SSC of all the cells. (D) Histogram of TER119 of CD45−CD11b−GR1− cells. (E) Plot of CD44 versus TER119 of the TER119 positive cells without gating. (F) Plot of CD44 versus TER119 of the TER119 positive cells with the gating on CD44hiTERlow population I. (G) Plot of CD44 versus FSC of the TER119 positive cells, showing naturally occurring clusters. (H) Plot of CD44 versus FSC of the TER119 positive cells with gating the population II, III, IV, V and VI. (I) Representative cytospin images of the sorted populations. (J) Histogram of FSC of distinct stage of erythroblasts. (K) Proportion of distinct stage of erythroblasts in bone marrow. Jing Liu et al. Blood 2013;121:e43-e49 ©2013 by American Society of Hematology

Representative erythropoiesis profiles of bone marrow and spleen of 4 Representative erythropoiesis profiles of bone marrow and spleen of 4.1R−/− and phlebotomy-induced mice. Representative erythropoiesis profiles of bone marrow and spleen of 4.1R−/− and phlebotomy-induced mice. (Top panel) Representative erythropoiesis profiles of bone marrow. Note that there is an increased proportion of nucleated erythroblasts (I+II+III+IV) which is accompanied by a decreased proportion of enucleated erythrocytes (V+VI) in both 4.1R−/− and phlebotomy-induced WT bone marrow compared with WT bone marrow. (Bottom panel) Representative erythropoiesis profiles of spleen. Note that there are very few erythroblasts in WT spleen but a large proportion of nucleated erythroblasts in the spleens of both 4.1R−/− and phlebotomy-induced WT anemic mice. Jing Liu et al. Blood 2013;121:e43-e49 ©2013 by American Society of Hematology

Altered terminal erythroid differentiation of Hbbth1/th1 mice and normalization after thansferrin therapy. Altered terminal erythroid differentiation of Hbbth1/th1 mice and normalization after thansferrin therapy. (A) Representative erythropoiesis profiles of bone marrow and spleen of Hbbth1/th1 mice. (Top panel) The representative erythropoiesis profiles of bone marrow. A decrease in fraction I (proerythroblast) and fraction VI (mature red cells) is clearly seen in PBS-treated thalassemia bone marrow and is restored in transferrin-treated bone marrow. (Bottom panel) Representative erythropoiesis profiles of spleen. There are very few erythroblasts in WT spleen, but the spleen of thalassemia mouse is highly erythropoietic and is restored in transferrin-treated spleen. (B) Western blot revealed an increase in soluble serum TfR1 in Hbbth1/th1 relative to WT mice and is normalized after transferrin treatment. Jing Liu et al. Blood 2013;121:e43-e49 ©2013 by American Society of Hematology