Fluorescent nanosensor based flux analysis: Overview and the example of glucose

Many metabolic events occur either too fast or within specifi c cells in a complex tissue, making them beyond the reach of standard isotope-based tracer methods. In order to discover and investigate such phenomena, our group has recently developed a technology that measures the rate of glycolysis in single cells with a temporal resolution of seconds. This GLUT-block method combines real-time intracellular glucose measurements with a FRET nanosensor and a blocker of the plasma membrane glucose transporter GLUT. This chapter contains an explanation of the experimental procedures and a description of the necessary equipment, including mention of cost options. Mathematical modeling of cellular glucose dynamics and experimental data are used to illustrate the possibilities and limitations of the method. Possible practical problems are discussed together with strategies to circumvent them.