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 Ca2+ 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.
Yamada
Katsuya
K
Department of Physiology, Hirosaki University School of Medicine, Aomori 036-8562, Japan.
Saito
Mikako
M
Matsuoka
Hideaki
H
Inagaki
Nobuya
N
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence
D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose