Quantifying Glucose Uptake in INS-1 Cells

A fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG) (Invitrogen #N13195), was used to measure glucose uptake in INS-1 cells according to the manufactory's instructions. Briefly, a 100 µM of 2-NBDG (dissolved in ethanol) was added over the transfected cells (as described above) (100 µM/1 ml medium/well) and incubated for one hour at 37 °C. Next, trypsin harvested cells were washed twice with cold-PBS, then centrifuged for 5 min at 1500 rpm at 4 °C. The supernatant was aspirated and cell suspended in 200 µl of cold-PBS in each tube. Cells were immediately analyzed and quantified by flow cytometry (BD FACS Aria™ III) at excitation 465 nm and emission at 540 nm .