2-NBDG as a marker for detecting glucose uptake in reactive astrocytes exposed to oxygen-glucose deprivation in vitro.

Abstract

Glucose is a necessary source of energy for sustaining cell activities and homeostasis in the brain. Enhanced glucose uptake protects cells during energy depletion including brain ischemia. Astrocytes enhance their glucose uptake during ischemia to supply substrates to neurons and thus support neuronal survival. Radiolabeled substrates are commonly used for in vitro measurement of glucose uptake in astrocytes. Here we optimized a method to measure glucose uptake by astrocytes during oxygen-glucose deprivation (OGD) using the fluorescent substrate 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Uptake buffers for 2-NBDG were the same as for (14)C-labeled α-methyl-D-glucopyranoside. Cell lysis buffer was optimized for observing the fluorescence of 2-NBDG, and Hoechst 33258 DNA staining was used for normalization of the 2-NBDG concentration. Uptake was performed on cultures of primary astrocytes by incubating the cells at 37 °C in buffer containing 25-200 μM 2-NBDG. Flow cytometry was performed to visualize uptake in intact cells, and a fluorescence microplate reader was used to measure the intracellular concentration of 2-NBDG in cell homogenates. 2-NBDG uptake was concentration dependent in astrocytes that were exposed or not exposed to OGD. OGD significantly increased 2-NBDG uptake by about 1.2 to 2.5 times in astrocytes compared to control cells. These results show that 2-NBDG can be used to detect glucose transport in astrocytes exposed to OGD.