Cytometry Workshops

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Core Faculty

Dr. T. Vincent Shankey

Dr. T. Vincent Shankey received his Ph.D. degree in Immunology and Medical Microbiology from the University Of Florida School Of Medicine (1977), and was a postdoctoral fellow at the University of Pennsylvania from 1977 to 1981. His research has used flow and image cytometry for over thirty years, working in clinical flow cytometry for much of that time. Before joining the Advanced Technology Center/Systems Research Group at Beckman Coulter in 2001, he was the Director of Research for the Urology Department and Scientific Director of the Clinical Flow Cytometry laboratory at Loyola University Medical Center near Chicago, Illinois.

Lab Module

Signal transduction pathways represent the major communications networks that transmit signals from multiple cell surface receptors to the nucleus, activating specific gene targets. These pathways serve an important function in integrating signals from individual pathways, to regulate cell death, or survival, cell proliferation and cell differentiation. Flow cytometric techniques have been developed to measure specific signaling pathways in the context of cell surface immunophenotypic analysis. In AML patients, we have studied the impact of several targeted agents on the MAPK, STAT, and PI3K pathways, in addition to monitoring phosphorylation of the ribosomal S6 protein. These approaches provide a unique insight into the biologic heterogeneity of human leukemias, and offer the potential to monitor individual patients' responses to targeted agents, including the potential for "real time" drug dose monitoring. LPS is known to activate multiple signaling pathways in peripheral blood monocytes via the TLR4 receptor complex. Following LPS exposure in vitro, monocytes rapidly activate all three major MAP Kinases (ERK, p38, and SAP/JNK), in addition to PI3 Kinase and IKK/NF?B pathways. Flow cytometry provides a unique methodology to monitor these signaling pathways, allowing a coordinated measurement of the quantity of multiple phospho-epitopes in the context of cell surface plus other cellular markers. In this module, we will perform an LPS stimulation assay of fresh peripheral blood monocytes, and will demonstrate the kinetics of activation and dephosphorylation of different pathways.

Relevant Literature

Jacobberger JW, Sramkoski RM, Frisa PS, Ye PP, Gottlieb MA, Hedley DW, Shankey TV, Smith BL, Paniagua M, and Goolsby CL. Immunoreactivity of STAT5 phosphorylated on tyrosine 694 as a cell-based measure of Bcr/Abl kinase activity. Cytometry 54A;75-88, 2003.

Chow, S., Hedley D, Grom P, Magarri R, Jacobberger, and T.V. Shankey. A whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of phospho-epitope expression in leukocyte subpopulations. Cytometry 67A;4-17, 2005.

Chow, S., D. Hedley, and T. V. Shankey. Measurement of Oncogenic Signaling Proteins by Flow Cytometry. Current Protocols in Cytometry 9:27, 2008.

Hedley D.W., S. Chow , C. Goolsby, and T.V. Shankey. Pharmacodynamic Monitoring of Molecular-Targeted Agents in the Peripheral Blood of Leukemic Patients using Flow Cytometry. Toxicol Pathol. 36:133-139, 2008.

Woost, P.G., L.A. Solchaga, H.J. Meyerson, T.V. Shankey, C.L. Goolsby, And J.W. Jacobberger. High Resolution Kinetics of Cytokine Signaling in Human CD34/CD117-Positive Cells in Unfractionated Bone Marrow. Blood (accepted for publication) 2010.