Multiparameter flow cytometric assay to monitor the activation of cytokine signaling pathways in specific subsets of primary erythroid cells

BIOT 99

Weijia Wang, weijia.wang@utoronto.ca and Julie Audet, julie.audet@utoronto.ca. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, 11th floor, Toronto, ON M5S 3E1, Canada
Cytokine synergism is a common theme in tissue and cellular engineering. However, an understanding of the cellular and population mechanisms at play is often lacking. Stem Cell Factor (SCF) and Erythropoietin (EPO) have a synergistic effect on erythroid cell output in cultures of bone marrow cells. Previous studies have demonstrated that these cytokine effects were mainly sequential, with each cytokine acting on different cell populations. We are now interested in determining if a specific cell subset can integrate both SCF and EPO signals during erythroid development. Since such study requires single-cell measurements and the ability to distinguish different cell populations, we have developed a flow cytometric assay that enables the simultaneous detection and quantitation of intracellular phospho-proteins (ERK and STAT5) and cell-surface markers (c-kit and CD-71). A better understanding of the mechanisms underlying the synergistic interaction between EPO and SCF can facilitate the large-scale production of erythrocytes for transfusion.