A real-time method of imaging glucose uptake in single, living mammalian cells

This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D -glucose derivative, 2-[ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy- D -glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca 2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.