Many metabolic events occur either too fast or within specific 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.
Glycolysis GLUT Hexokinase FRET microscopy Nanosensor Real-time Cytochalasin B
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We thank past and present members of the Barros Laboratory for their contribution to the development and validation of this method and thank Karen Everett for critical reading of the manuscript. This work was partly supported by Fondecyt grants 1100936 and 1130095. The Centro de Estudios Científicos (CECs) is funded by the Chilean Government through the Centers of Excellence Basal Financing Program of CONICYT.
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