Introduction

This chapter reviews and discusses two different flow cytometric approaches to the study of cellular physiology (defined as studies of the dynamic state of a cell population). The first approach uses fixed and permeabilised cells to detect and quantify epitopes on important regulatory molecules as a function of cell cycle phase or stage in the cell death process at a single time point per sample. This approach is static and the time domain, and consequently the physiological state, is inferred. The second approach uses live, dying or dead cells in a native state that have been stained with fluorescent molecules that ‘report’ the physiological state. These measurements are dynamic and fluctuate in real time.

The discussion and examples herein are biased towards the signalling pathways in cancer cells that, in aggregate, define the oncogenic phenotype. Published studies of signalling pathways have increased steadily since the early 1980s, but advances that provide a more complete picture of pathways as interacting nodal networks are more recent. At present, the exact definition of a pathway/network is not very precise. The following is a definition within the context of this chapter: ‘the aggregate biochemical process by which some particular cell behaviour (phenotype) is regulated that includes all chemical changes in the “resting state” that affect the cell phenotype significantly when components of the pathway are modulated.’

Logically, all components within the cell are interconnected to some degree; therefore, modulating one component will probably have some effect on all others. If the regulatory pathways are a multidimensional set of interconnected reactions, one area can only be viewed in isolation by invoking an arbitrary cut-off where the magnitude of an effect on another reaction is deemed insignificant.