Flow Cytometry (FCM) Analysis and Fluorescence-Activated Cell Sorting (FACS) of Erythroid Cells

Abstract

To study the process of erythropoiesis, it is important to be able to isolate erythroid progenitors and erythroblasts at distinct stages of development. During the past decade, considerable progress has been made on the development of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) methods for the analysis and isolation of both murine and human erythroid cells at distinct stages of erythropoiesis, based on changes in the expression of cell surface markers. A method for the identification of murine BFU-E and CFU-E cells was reported by Flygare et al., by negative selection for Ter119, B220, Mac-1, CD3, Gr1, Sca-1, CD16/CD32, CD41, and CD34 cells, followed by separation based on the expression levels of CD71. We developed an alternative method in which Ter119 is used as an erythroid lineage marker, and in conjunction with CD44 and cell size as differentiation markers, it is possible to unambiguously distinguish erythroblasts at each developmental stage during murine terminal erythroid differentiation. We also developed methods for the analysis and isolation of human erythroid cells at all developmental stages. BFU-E and CFU-E are characterized by CD45⁺GPA⁻IL-3R⁻CD34⁺CD36⁻CD71^low and CD45⁺GPA⁻IL-3R⁻CD34⁻CD36⁺CD71^high phenotypes, respectively; the combination of GPA, band 3 and α4-integrin are used to isolate erythroid cells at all of the terminal stages of human erythropoiesis, including proerythroblasts, early basophilic, late basophilic, polychromatic and orthochromatic erythroblasts.