PHOSPHO FLOW CYTOMETRY: SINGLE-CELL SIGNALING NETWORKS IN NEXT-GENERATION DRUG DISCOVERY AND PATIENT STRATIFICATION - Flow Cytometry in Drug Discovery and Development - page 293

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PHOSPHO FLOW CYTOMETRY:
SINGLE-CELL SIGNALING
NETWORKS IN NEXT-GENERATION
DRUG DISCOVERY AND PATIENT
STRATIFICATION

P ETER O. K RUTZIK ,S EAN C. B ENDALL ,M ATTHEW B. H ALE ,
J ONATHAN M. I RISH , AND G ARRY P. N OLAN

15.1 INTRODUCTION

Flow cytometry is a unique platform for cellular analysis given its ability to measure
the level of 10 or more antigens per cell, including cell surface markers, cell cycle
proteins, calcium levels, DNA content, and intracellular cytokines, at the rate of up to
5000 cells per second. Recently, phosphoproteins have been added to the list of
molecules that can be measured by flow cytometry with a method called phospho-
specific flow cytometry or phospho flow (see Refs [1-5] for reviews). In phospho flow,
antibodies that are specific only to the phosphorylated form of intracellular signaling
proteins are used to measure the activities of various kinases and their target proteins,
often transcription factors [6]. By simultaneously measuring phosphoprotein levels
alongwith levels of other antigens, such as cell surface markers, phospho flow enables
complex biochemical events to be measured in heterogeneous populations of cells
such as peripheral blood and immunological tissues. This has led our group to apply
phospho flow to all stages of drug discovery, from understanding disease mechanism
and initial target identification, through high-throughput drug screening and pre-
clinical testing, to patient stratification and diagnostics. A major benefit of phospho