Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

Abstract

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis

“…A total of 20,000 events were collected and analyzed using FlowJo software version 7.6.5. Erythroid lineage was assessed using Ter119 APC/CD71 PE markers combined with the forward scatter properties (26). Hematopoietic stem/progenitor cells were differentiated using SLAM markers (CD150 PE/CD48 APC), Sca-1 FITC (Ly6A-E), c-Kit Percp Cy5.5 (CD117), CD90 PE (Thy-1), and APC-tagged lineage mixture comprising of antibodies against CD3, B220 (CD45R), Ly6G and Ly6C (Gr-1), CD11b (Mac-1), and TER119.…”

-thalassemias: paradigmatic diseases for scientific discoveries and development of innovative therapies

“…71 The different stages of differentiation post CFUe were identified as proerythroblasts, basophilic, polychromatic, orthochromatic erythroblasts, reticulocytes and red blood cells. [71][72][73] Upon the binding of erythropoietin to EPO receptor (EPOR), the tyrosine kinase/Janus kinase 2 (JAK2) is phosphorylated, which in turn activates multiple signal transduction pathways crucial in erythropoiesis ( Figure 3). One such pathway consists of activation of Signal Transducer and Activator of Transcription 5 (Stat5) and downstream antiapoptotic B-cell lymphoma-extra large (BclxL) protein.…”

A Stochastic Model for Highway Accident Predictions with Winter Data

“…To know the existent of erythropoiesis we examined the expression of TER-119 antigen as a specific marker of erythroid-lineage [15][16][17]. In this experiment we showed that the number of TER-119 + cells in the bone marrow of CD122 -/-decreased before the manipulation with regulatory T cells.…”